The BiosearchTech Blog

Pipetting for qPCR

A "Referral from the Doctor" Blog Article-

Research trends in laboratories today increasingly steer towards gene expression analysis and genetic testing, often in the form of qPCR. As reproducibility is essential to genetic research it is imperative that scientists know the fundamentals of micro-volume pipetting.

Forward and Reverse Pipetting: This discussion is limited to the use of manual pipettors. Electronic pipettors are capable of other pipetting techniques such as dispensing, sequential dispensing and diluting which are not discussed here.

• Forward pipetting is used for aqueous solutions such as water, buffers, diluted saline, diluted acid or base. Appropriate aqueous solutions may also contain low concentrations of proteins or detergents. This technique is appropriate for milliliter and microliter volumes. In forward pipetting, aspiration involves compression of the key to the first stop followed by the slow release of the key, creating a vacuum within the barrel and aspirating the solution volume desired. Expulsion involves dispensing the solution by pressing the key down to and beyond the first stop to "blow out" the entirety of the aspirated volume.

• Reverse pipetting is used for viscous solutions, solutions with high vapor pressure or extremely small microliter volumes. In reverse pipetting, aspiration involves compression of the key to the second stop followed by the slow release of the key, creating a vacuum within the barrel and the aspiration of a volume greater than that selected. Expulsion involves pressing the key down to the first stop only, thus dispensing only the desired volume.

Pipetting micro-volumes:

• Pipette with smooth and deliberate action.
• Hold the pipette vertically at all times. This is best accomplished by using your index finger to dispense and aspirate instead of your thumb. 
• Immerse the pipette tip only slightly to avoid coating the outside of the tip with excess liquid that may be inadvertently transferred during dispensing.
• Pipette the initial volume directly to the bottom of the receiving container while lifting the pipette upward slowly so as not to introduce bubbles to the dispensed solution. Add additional volumes to the initial volume using the same technique.
  

Tips to improve accuracy:

• Pre-wet the tip. When pipetting greater than 10 microliters, it is good practice to do an initial aspiration and expulsion to decrease the amount of volume that will "stick" to the inside of the tip during dispensing. Pre-wetting will coat the inside of the tip and decrease the amount of volume lost by sample manipulation.
• Take sample temperature into consideration. When pipetting cold samples, the first aspiration is larger than all subsequent pipetting using the same tip. The opposite occurs when pipetting solutions warmer than ambient temperature wherein the first aspiration has a decreased volume than subsequent volumes using the same tip. To avoid these temperature related issues, always pre-wet the pipette tip before dispensing. 
• Consider the ambient temperature for your pipette. Pipettes are calibrated at room temperature. Using a pipette in a cold room will result in variable volumes being dispensed. 
• Use an appropriate pipette for the desired dispensed volume. Check the range of volumes allowed for your pipette and do not go over or below that recommended volume. For example, use a P2 pipette to dispense a 1 microliter volume as its range in linear between 0.2 through 2 microliters. Do not use a p20 which has a linear range between 2 through 20 microliters, for dispensing a 1 microliter volume. 
• Use the largest volume possible to make your dilutions. It is easier to maintain accuracy when pipetting a larger volume than a smaller volume. 
• Wait until the total volume has aspired before moving forward. It takes time to aspire and dispense volumes. One second should be allowed for the liquid to fill the tip.
• Account for the angle of inclination. The pipette should be held vertically at all times. During aspiration the hydrostatic pressure of the liquid decreases as the angle of inclination increases resulting in the over-aspiration or too much volume. The technique of pipetting at a 45 to 60 degree angle is appropriate for sterile technique pipetting associated most commonly with cell culture. Small deviations in the volumes associated with cell culture are not critical to the success of cell growth making the sterile technique preferable in that situation.
  

Posture:

• Maintains the natural curve of the spine. Prior to pipetting, stand or sit with your shoulders back, slightly, and parallel to the floor. Hold your head in the midline position and level so that your ears are directly over your shoulders and your eyes facing front. When standing, the pelvis should be shifted forward to align the hips directly over the ankles.
• Keep all objects close to the body and placed within an easy reach between usages. Minimize twisting and bending motions at the hip. 
• Lift objects that need to remain level or are awkward or heavy, with your back straight and using your legs. 
• Stretch your hands and arms frequently. Grip the pipette gently and use only the minimum force needed to activate the plunger. Alternate hands used to pipette, if possible. Purchase pipette tips that do not require high insertion force.
  

Pipette Maintenance:

• Recalibrate every 6-12 months, depending on usage. Service should include recalibration, replacement of seals and greasing of all moving parts.
• Inspect the pipette for damage or discoloration before each use. Any compromise to the pipette condition may have significant repercussions in pipetting reproducibility. Have the pipette serviced immediately to avoid wasted time and money on failed experiments due to pipetting errors. 
• Clean the pipette before each daily use. Wipe down the barrel, handle, aspirator key and plunger release key with a nucleotide-free 0.1 N HCl solution using laboratory grade tissue paper, such as Kimwipes, to destroy any RNA or DNA on the surface of the pipette. Follow that wash with a nucleotide-free 70% Ethanol solution to precipitate and remove the lingering nucleotide fragments. This same procedure is excellent for cleaning your work station and associated equipment.
• Store pipettes in an upright position using a pipette stand or hanging pipette holder. Pipettes have a hollow barrel and so may easily become contaminated with liquid remaining in the tip if placed on their sides. By keeping pipettes upright and using filtered pipette tips, the pipette barrel is less likely to become contaminated.
• Use tips designed for the pipette. Each manufacturer has a recommended tip to be used with the pipette. Only use tips that fit the specifications listed in the brochure or user manual for your specific pipette brand and type.

By following the guidelines suggested above individual scientists may improve their pipetting accuracy and consequentially the reproducibility of data. For more information on pipetting technique go to www.rainin.com or www.eppendorf.com.

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist 

Molecular Beacons - Lights in the storm

A "Referral from the Doctor" Blog Article-

Molecular Beacons are a special type of dual-labeled oligonucleotide probe. Beacons are hairpin loop structures with a 5'-fluorophore and a 3'-quencher dye. The stem region is a short sequence of 5-7 complementary bases. The loop sequence is complementary to the target sequence. In the absence of target or prior to amplification, the stem anneals to form a closed hairpin conformation which holds the reporter and quencher close together to enable efficient FRET quenching and to promote contact or static quenching. The beacon is engineered such that the probe-target hybrid is more stable than the closed hairpin conformation, which is respectively more stable than a probe-mismatch hybrid. Therefore it is only in the event of a perfect-match hybridization that signal occurs, allowing beacons to discriminate mismatches as small as a single nucleotide polymorphism (SNP).

Structure

Q: What parts are similar to dual-labeled Black Hole Quencher® (BHQ) probes?
A: A 5'-fluorophore and a 3'-BHQ dye covalently bound to the oligonucleotide termini.

Q: What parts are unique?
A: A target-binding region comprised of a stem-loop structure resembling a "closed hairpin".

Q: What is its native conformation at melting and annealing temperatures?
A: Melted - random coil conformation
Annealed (perfect match) - stable double helix, probe-target hybrid (high signal to noise ratio)
Annealed (mismatch) - hairpin conformation (FRET quenched, no signal)

Mechanism

Q: How does it yield signal?
A: The target-binding region is located within the loop portion of the hairpin conformation and sets atop an annealed stem region. When a perfectly matched complementary sequence is available, the probe region hybridizes to the complementary sequence. Conformational changes associated with hybridization force the hairpin stem region open, separating the fluorophore and quencher, decreasing FRET quenching and releasing fluorescence.

Q: What happens with each consecutive PCR cycle?
A: 1. At high temperatures target DNA duplexes and Molecular Beacons are melted and maintain random coil conformations.
2. As the reaction cools to temperatures appropriate for the binding of primers and targets, the beacon will reach an equilibrium between the hairpin and hybridized states. This equilibrium depends on the availability of a perfect-match target, whose binding by the probe is thermodynamically favored, by design, over the hairpin conformation. This ensures that the molecular beacon can hybridize when appropriate target is available. In the hybridized conformation the reporter and quencher dyes are separated, releasing fluorescence.
3. During polymerization, the beacon is dislodged from the target sequence, refolds into and maintains the hairpin conformation until the next cycle of amplification. The increase in fluorescence intensity with repeated PCR cycles indicates the accumulation of product and allows for accurate quantification of template.

Q: What if the available target is a mismatch?
A: Mismatched hybrids are less stable than reformation of the hairpin stem. Therefore signal is only produced when the target binding region hybridizes to a perfect-match target sequence. This enables specificity of signal generation to be accurate at the level of single nucleotide polymorphisms (SNP).

• High signal to noise ratio - the efficiency of FRET quenching, when in the unhybridized state, decreases errant fluorescence and enables a high signal to noise ratio with hybridization.
• High specificity - single base mismatches can be detected.
• Post-PCR melt curve analyses - Molecular Beacons do not require the enzymatic activity of a polymerase for fluorescent signal generation in qPCR and, under non-hydrolytic conditions, allow for post-PCR melt curve analyses.
Applications: Single and multiplexed, quantitative and qualitative, real-time and endpoint qPCR analyses; allelic discrimination; SNP analysis; DNA microarray-immobilized probes and biosensors and; as nuclease-resistant antisense probes for detection RNA in vivo.

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist

Tyagi, S. and Kramer, F.R. "Molecular beacons: probes that fluoresce upon hybridization". Nature Biotechnology 14 (1996): 303-308.
Bonnet, G., Tyagi, S., Libchaber, A., Kramer, F.R. "Thermodynamic basis of the enhanced specificity of structured DNA probes". Proc Natl Acad Sci. 96 (1999): 6171-6176.
Marras, S.A.E., Kramer, F.R., Tyagi, S. "Multiplex detection of single-nucleotide variations using molecular beacons". Genet Anal, 14 (1999): 151-156.
Marras, S.A.E., Kramer F.R., and Tyagi, S. "Genotyping single nucleotide polymorphisms with molecular beacons". Single nucleotide polymorphisms: methods and protocols (v. 212). Ed. P.Y. Kwok. Totowa, NJ: The Humana Press, 2003. 111-128.
Vet, J.A.M. and Marras, S.A.E. "Design and optimization of molecular beacon real-time polymerase chain reaction assays". Oligonucleotide synthesis: Methods and Applications (v. 288). Ed. P. Herdewijn. Totowa, NJ: Humana Press, 2004. 273-290.
Bustin, S.A. A-Z of Quantitative PCR. (IUL Biotechnology Series). La Jolla, California: International University Line, 2004.
Broude, Natalia E. "Molecular Beacons and Other Hairpin Probes". Encyclopedia of Diagnostic Genomics and Proteomics. (2005): DOI: 10.1081/E-EDGP 120020717. 846-850
Public Health Research Institute. "Designing Molecular Beacons". www.molecular-beacons.org

 

Dual-labeled BHQ probes - Performance to “dye” for

A "Referral from the Doctor" Blog Article-  

A BHQ® probe is a dual-labeled oligonucleotide covalently labeled with a fluorophore and a Black Hole Quencher® (BHQ) dye.

Q. What are the main components of a dual-labeled Black Hole Quencher® (BHQ) probe?

• An oligonucleotide, typically 30 bases long
• A 3' BHQ dye
• A 5'-fluorophore (reporter) dye

Q. What is its native conformation at melting and annealing temperatures?
Melted - random coil conformation
Unhybridized - unrestricted hairpin, FRET-quenched (no signal)
Hybridized- stable double helix, probe-target hybrid (signal)

Q. How does it yield signal?
When a complementary sequence is available, the probe hybridizes to the complementary sequence. Conformational changes associated with hybridization separate the fluorophore and quencher, decreasing FRET quenching and releasing fluorescence.
Q. What happens with each consecutive PCR cycle?
1. Heat melts or denatures the probe, sense and antisense strands of a DNA duplex.
2. As temperatures cool, hydrophobicity and electrostatics promote dye-dye attractions and enhance fluorescent quenching.
3. At annealing temperatures, the primers and the BHQ probe anneal to their complementary sequences within the target DNA. Conformational changes during hybridization separate the dyes which decreases FRET quenching thus releasing fluorescence.
4. During elongation, the DNA polymerase incorporates nucleotides complementary to the strand as it progresses in a 5' to 3' direction from the primer. When the polymerase encounters the 5'-end of the probe, it cleaves off the nucleotide, or a flap of nucleotides, with the bound reporter dye, thereby permanently separating the reporter and quencher dyes.

.

Dual-labeled BHQ probes have replaced earlier reporter-quencher dye pairings, such as FAM-TAMRA or FAM-DABCYL. In such sub-optimal probes, the quencher has inherent limitations such as auto-fluorescence or insufficient quenching at certain wavelengths which limit the choice of quenchable fluorophores. In contrast, the BHQ dyes:

• are highly efficient dark quenchers;
• have broad absorption spectra;
• and yield high signal to noise ratios.

BHQ dyes can be paired with all common reporter dyes emitting between the ultraviolet and infrared wavelengths, thereby making multiplexed hybridization assays easy to design and interpret.
Applications: Single and multiplex, quantitative and qualitative, real-time and endpoint PCR analyses; allelic discrimination; and SNP detection.

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist

Didenko, V.V. "DNA probes using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications". BioTechniques 31 (2001): 1106-1121. (Review)
Bustin, S.A. A-Z of Quantitative PCR. (IUL Biotechnology Series). La Jolla, California: International University Line, 2004.
Ranasinghe, R.T., Brown, T. "Fluorescence based strategies for genetic analysis". Chem. Commun. (2005): 5487-5502.
Johansson, M.K. "Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers." Methods in Molecular Biology (v. 335, ch. 2). Ed. V.V. Didenko. Totowa, NJ: Humana Press, 2004. 17-29.
Marras, S.A.E. "Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols" Methods in Molecular Biology (v. 335, ch. 7). Ed. V.V. Didenko. Totowa, NJ: Humana Press, 2004. 3-16.
Biosearch Technologies website. Black Hole Quencher Dyes, www.biosearchtech.com/support/applications/dyes-from-biosearch-technologies
Biosearch Technologies website. Genotyping-qPCR, www.biosearchtech.com/support/applications/genotyping-qpcr

This Year's MVP (Most Valuable Practice): Touchdown PCR

A "Referral from the Doctor" Blog Article-  

The aim of the Polymerase Chain Reaction (PCR) method is to amplify or increase the number of copies of a target sequence. Most often that sequence is part of a gene of interest and the total copy number or relative copy number of that transcript will impart some knowledge to the researcher regarding the impact of a drug, disease status of a sample or may represent a control condition. In some situations, the copy numbers of a particular transcript are very low and it may be necessary to nudge the PCR reaction in favor of amplifying the exact splice variant, family member or a construct the researcher wishes to examine.

 

 

In PCR, the temperature at which primers anneal during a cycle determines the specificity of annealing. The melting point (T_m) of the coolest primer sets the upper limit on annealing temperature. At temperatures just below the T_m, only very specific base pairing between the primer and the template will occur. As the temperature decreases, primer binding becomes less specific. Non-specific primer binding results in the amplification of undesired products and may mask the actual copy number of the gene of interest.

Touchdown PCR (Step-down PCR) is a variant of qPCR that reduces nonspecific target amplifications by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5 °C) above the T_m of the primers used. This ensures high specificity binding of the primers and is least-permissive of non-specific binding. As the PCR continues, each cycle occurs at lower and lower temperatures, decreasing by 0.2 °C per cycle. At the later cycles, it is a few degrees (3-5 °C) below the primer T_m. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles. Thus, the first sequence amplified is the one between the regions of greatest primer specificity and will be the most abundant product after cycling is complete.

This method is effective for amplification of one or only a few gene transcripts at a time. For larger comparative studies, standard PCR thermocycling conditions are recommended.

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist

Hecker K, Roux K (1996). "High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR". Biotechniques 20 (3): 478-85. PMID 8679209

An Alternative to DNAse I heat inactivation: LiCl precipitation

A "Referral from the Doctor" Blog Article-

Genomic DNA contamination has been a hot topic for qPCR researchers since the beginning. It is impossible to remove all DNA from RNA preparations without additional steps. The most commonly used technique is to incubate with DNAse I. The deoxyribonuclease I (DNAse I) enzyme catalyzes the hydrolytic cleavage of phosphodiester linkages adjacent to a pyrimidine nucleotide. It acts on single-stranded DNA, double-stranded DNA, and chromatin. DNAse I activity will degrade trace amounts of genomic DNA (up to 10 µg/mL) that could otherwise result in falsely positive signals in subsequent RT-PCR reactions. DNAse I is a heat-inactivated nuclease, requiring both the presence of EDTA and temperatures of 75^oC for 5 minutes for complete inactivation. The extreme temperatures associated with heat-inactivation of the enzyme may cause damage to the RNA through chemical mediated degradation if even small amounts of metal ions are present. Lower temperatures will not fully inactivate the DNAse before reverse transcription of the RNA to cDNA. Because we cannot discriminate between cDNA and DNA amplification products, initial copy number determination is compromised.

While acid phenol extraction has been a reliable alternative method for DNA removal from nucleic acid preparations, the formation of an organic phase and the resultant hazards associated with working with organic wastes, has made this technique obsolete. Other techniques such as using immobilized DNAses or the use of manganese instead of magnesium in the DNAse I incubation have contributed significantly to solving the issue of genomic DNA carryover contamination.

The little known alternative is to reduce contaminating genomic DNA with a Lithium Chloride (LiCl) precipitation (ppt). LiCl ppt can be used after DNAse I treatment to inactivate the enzyme without using heat-inactivation. LiCl ppt will selectively precipitate full-length RNA transcripts from solution to the exclusion of protein or DNA and is effective at removing free nucleotides. The LiCl ppt method requires only low concentrations, 2.5M, of LiCl and short, 30 minute, room temperature incubations. RNA recovery from the precipitant solution is dependent upon centrifugation conditions. Centrifugation at high speeds around 16K x g, for 20 minutes at 40C will enable the recovery of as little as 50ng of RNA. For an inexpensive, reproducible, efficient way to purify your RNA after isolation and treatment with DNAse I, try the Lithium Chloride precipitation method.

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist  

References:
Z. Huang, M.J. Fasco, L.S. Kaminsky. Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR. Biotechniques (1996) 20(6): 1012-1020
B.L. Ziegler, C. Lamping, S. Thoma, C.A. Thomas. Single-cell cDNA-PCR: removal of contaminating genomic DNA from total RNA using immobilized DNAse I. Biotechniques (1992) 13(5): 726-729
P. Bauer, A. Rolfs, V. Regitz-Zagrosek, A. Hildebrandt, E. Fleck. Use of manganese in RT-PCR eliminates PCR artifacts resulting from DNAse I digestion. Biotechniques (1997) 22(6): 1128-1132
O.P, Das, C. Alvarez, M. J. Chaudhuris. Molecular methods for genetic analysis of
maize. Methods Mol Cell Biol. (1990) 1: 213-222
J. Sambrook, and D.W. Russel. "Protocol 16: Removal of small fragments of nucleic acid from preparations of plasmid DNA by precipitation with lithium chloride". Molecular Cloning: A laboratory manual, 3rd Edition, Volume 1. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York. 2001. 82-83.

Slope Efficiency Equals Manageable and Corrected PCR

A "Referral from the Doctor" Blog Article-  

ε = m.c.2 (shall I explain? slope efficiency equals manageable and corrected PCR)

IF	m = manageable dilutions of standard sequence, ideally 10 fold serial dilutions of a plasmid containing known copy numbers of the gene of interest. Copy number of your plasmid control gene is calculated as: 9 x 1012 molecules / Kb = n / μg DNA
Assuming	c = corrected data means you have determined the actual copy number in your serial dilutions of plasmid by back calculating from the acquired Ct value, assuming the highest concentration is correct.
AND	"2" or ln = is the natural log of a number or based 2 log. As PCR is a function of doubling the copy number with each cycle, all cycles to threshold (Ct) numbers may be compared using a natural log function: 2(delta Ct) or 2(ave Ct of standard 1 - ave Ct of standard 2)
THEN	ε = calculated efficiency of the slope would ideally equal 1, meaning 100% ε =[10(-1/slope)] - 1

Note: When creating standard curves for the purpose of quantitative analysis of real-time RT-PCR, it is essential that the efficiency of the amplification be greater than 88% for all genes analyzed. When making direct comparisons the efficiencies must also be within 5% of each other. The "r" value for each slope must be better than 0.95. A variability of 5% between technical replicates of the standard curve is typical, as is a 10% difference in efficiency between the standard curves of different genes run on the same plate.

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist  

References:
C.A. Heid, J. Stevens, K. J. Livak, P. M. Williams. Real Time Quantitative PCR. Genome Methods (1996) 6:986-994
J. Winer, C.K.S. Jung, I. Shackel, P. M. Williams. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Analytical Biochemistry (1999) 270:41-49
R. Higuchi, C. Fockler, G. Dollinger, R. Watson. Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. Biotechnology (1993) 11(9):1026-1030
K.J. Livak, T.D. Schmittgen. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods (2001), 25: 402 - 408
M.W.Pfaffl. A new mathematical model for relative quantification in real-time RT-PCR. Nucl. Acids Res. (2001) 29:2002-2007
U.E. Gibson, C.A. Heid, P.M. Williams. A novel method for real time quantitative RT-PCR. Genome Res. (1996) 6:995-1001

Bringing Back a Champion: Competitive Quantitative Real-time RT-PCR

A "Referral from the Doctor" Blog Article-

Competitive qRT-PCR has become a lost art. Early titans of probe-based PCR used this technique to minimize variability between samples and experiments due to differences in reverse transcription and amplification efficiency. This technique facilitates the absolute measurement of initial gene target copy number based on known copy numbers of a control template.

This is accomplished through the addition of an internal control, a competitor sequence to the mRNA target in the sample. The competitor sequence is similar enough to use the same primer pairs as the target sequence but has a unique central sequence allowing for analysis with a different probe and reporter. Plasmid DNA or in-vitro transcribed RNA is commonly used as a competitor sequence and standard for quantification. To detect the initial copy number of a gene of interest, multiple replicates of a fixed amount of the target mRNA are dispensed into tubes. In the same tubes, 10 fold serial dilutions of competitor RNA are "spiked" in sequentially. Using one set of primers and two fluorogenic probes labeled with different dyes, real-time PCR data can be collected for both sequences simultaneously.

To determine copy number, the mean Cycles to Threshold (CT) values from the target sequence and the mean CT values of the competitor serial dilutions are plotted on the y axis, against the known copy number of the competitor sequence on the x axis. The number of initial copies of the target gene can be calculated from the theoretical equivalence point, where the CT of the target gene equals the CT of the competitor sequence - the point of intersection for each linear plot. It is essential that the efficiency of both amplifications be greater than 88% and furthermore within 5% of each other for accurate analysis.

This method of quantitative PCR is returning to the forefront of gene expression analysis, particularly for low expressing genes or those difficult to amplify. Success in the modern age is dependent upon the equipment capability, the stringency of validation and selection of fluorogenic probes. For a catalog of available probe formats, visit: http://www.biosearchtech.com/products/fluorogenic-probes-and-primers

Thus, we can rise above and see beyond because we stand on the shoulders of giants.
-concept attributed to Bernard of Chartres

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist  

References:
H. Tani, T. Kanagawa, S. Kurata, T. Teramura, K. Nakamura, S. Tsuneda, N. Noda. Quantitative method for specific nucleic acid sequences using competitive polymerase chain reaction with an alternately binding probe. Anal. Chem. (2007) 79(3):974-979
E. Barbieri, G. Riccioni, A. Pisano, D. Sisti, S. Zeppa, D. Agostini, V. Stocchi. Competitive PCR for quantitation of a cytophaga-flexibacter-bacteroides phylum bacterium associated with the tuber borchiivittad. Mycelium. Applied and Environmental Microbiology (2002)68(12):6421-6424
C. Orlando, P. Pinzani, M. Pazzagli. Developments in Quantitative PCR.ClinChem Lab Med (1998) 36(5):255-269

The New Favorite Reference Gene: 36B4, a.k.a. RPLP0

A "Referral from the Doctor" Blog Article-

Ribosomes are comprised of two subunits, one small (40S) and one large (60S). The 36B4 gene encodes an acidic ribosomal phosphoprotein P0 (RPLP0), which forms tight associations with the smaller 40S protein (L12). 36B4 protein is part of a pentameric complex that forms a stem-like structure, protruding into the cytoplasm, off of the 60S subunit. This protrusion functions to support the GTPase steps in the translocation of protein synthesis. Why is 36B4 fast becoming the new reference gene for probe-based real time RT-PCR?

The 36B4 cDNA nucleotide sequence has highly conserved regions in the 5-prime end of its open reading frame that cross tissue and species boundaries. When compared to the transcript levels of other common reference genes, such as beta-actin and cyclophilin, 36B4 proved to be a very reliable and consistent standard for use in gene expression analysis among multiple tissues including: brain, heart, liver, kidney, muscle and lung. Furthermore, the nucleotide sequence homology between species is remarkably high. For canis familiaris (dog) the homology to the human sequence is 94%; for pan troglodytes (chimpanzee), 99%; and for bos taurus (bovine), 94%. In basic science the most commonly used species are mus musculus (mouse) and rattus norvegicus (rat) whose 36B4 maintains a homology with the human gene of 88% and 89% respectively. As with other highly expressed genes, 36B4 has 8 related pseudo-genes in the human genome, found on chromosomes 1-3, 11, 14, 15 and 18.

Recent trends have supported the use of multiple reference genes as normalizing factors in real-time RT-PCR. For a list of the most highly recommended reference genes, see our blog article, What Genes are Hot, What Genes are Not.

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist

References:

Rie Akamine , T. Yamamoto, M. Watanabe, N. Yamazaki, M. Kataoka, M. Ishikawa, T. Ooie, Y. Baba, Y. Shinohara. Usefulness of the 5' region of the cDNA encoding acidic ribosomal phosphoprotein P0 conserved among rats, mice and humans as a standard probe for gene expression analysis in different tissues and animal species. (2007) J. Biochem. Biophys. Methods 70: 481-486

Jorge Laborda. 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein P0. (1991) Nucleic Acids Research 19 (14): 3998
Key words: 36B4, RPLP0, acidic ribosomal phosphoprotein P0

What Genes are Hot, What Genes are Not

 A "Referral from the Doctor" Blog Article-

 Top 10 Reference Genes for probe-based Real-time PCR in 2009:

• Acidic Ribosomal Phosphoprotein P0 (RPLP0 or 36B4)
• ATP-synthase subunit 5B (A5B)
• Tumor Protein Translationally controlled 1 (TPT1)
• Signal Recognition Particle 14kDa (SRP14)
• TATA-Binding Protein (TBP)
• Eukaryotic Elongation Factor 1A1 (EEF1A1)
• Hypoxanthine Phosphoribosyl-Transferase 1 (HPRT1)
• poly-Ubiquitin (Ubi)
• Glyceraldehyde-3-phosphate dehydrogenase (G3PD)
• Beta-actin (ACTB)

See next week's blog for follow up information!

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist

References:
A. Pilbrow, L. Ellmers, M. Black, C. Moravec, W. Sweet, R. Troughton, A.M. Richards, C. Frampton, V. Cameron. Genomic selection of reference genes for real-time PCR in human myocardium. BMC Medical Genomics (2008) 1(1):64

Li-Yun Fu, Hu-Liang Jia, Qiong-Zhu Dong, Jin-Cai Wu, Yue Zhao, Hai-Jun Zhou, Ning Ren, Qin-Hai Ye, Lu-Xiu Qin. Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses. BMC Cancer (2009) 9:49

E.D. Silveira, M. Alves-Ferreira, L.A. Guimaraes, F. R. da Silva, V. T. de Campos Carneiro. Selection of reference genes for quantitative real-time PCR expression studies in apomictic and sexual grass Brachiaria brizantha. BMC Plant Biology (2009) 9:84

G. Silberberg, K. Baruch, R. Navon. Detection of stable reference genes for real-time PCR analysis in schizophrenia and bipolar disorder. Analytical Biochemistry (2009) 391(2):91-97

B. Etschmann, B. Wilcken, K. Stoevesand, A. von der Schulenburg, A. Sterner-Kock. Selection of reference genes for qualitative real-time PCR analysis in canine mammary tumors using geNorm algorithm. Vet Pathol. (2006) 43:934-942