Sunday, May 16, 2010

An Experimental Analysis of Major Factors Contributing to Unexpected Real Time RT-PCR Efficiency

Student: Ann L. Bohannon-Stewart
Class: Individual Studies Spring Semester 2010
Professor: Benny Washington

Real time reverse transcription polymerase chain reaction (RT-qPCR) assay is widely used in the determination of RNA expression in various tissues of various organisms. If properly validate, this convenient method can quantify specific RNA sequence in few hours, comparing with days needed for Northern blot analysis. RT-qPCR assay is commonly performed either in two steps or one step. In many reports, one step RT-qPCR using SYBR green chemistry is the method of choice. Despite the widespread application, the extent to which RT-qPCR data are unreliable and the resulting biological validity are under-appreciated and not well acknowledged. Many gene expression studies using RT-qPCR method convert gene expression level with 2-ΔΔCt. While this conversion is convenient and appropriate when amplification efficiency is close 100%, problems arise when efficiency significantly deviates from the presumptive value. Using SYBR green one step RT-qPCR kit, sometimes RT-qPCR efficiency may appear to be greater than 400%. It is necessary to determine the cause of the appeared high efficiency and to develop approaches to overcome the problem. The major factors contributing to the efficiency variations are experimentally tested in the current study. Primers for chicken CD8α mRNA were used to determine how reagent and different parameters in RT and PCR stages affect the efficiency of RT-qPCR assay.

The efficiency of RT-qPCR may be affected by many factors, including template and primer concentrations, specific template sequences, reverse transcriptases, DNA polymerases and other components of the reaction system. In addition, sporadic factors such as inactivated enzymes, inaccuracy of pipetting and human errors also contribute to problems of amplification efficiency. In this study, we tested how some of these factors work. First we examined PCR efficiency using DNA template under RT-qPCR chemistry. We then examined the effect of primer concentrations on RT-qPCR efficiencies. Third, we tested how different primers behave under similar conditions. We have also carefully controlled experimental procedures try to minimize the human errors so that experiment become more reliable. This study has provided valuable information for future RT-qPCR assay design and quality control in gene expression studies.

Materials and Methods:
DNA template was generated by RT-PCR amplification of chicken CD8a mRNA with gene-specific primers, purified with gel electrophoresis and gel extraction kit (Qiagen). Serial dilutions of the DNA template were made for high copy number range (1.22 x 10^19, 6.08 x 10^18, 3.04 x 10^18 copies/µl) and low copy number range (1.2 x10^7, 3.0x10^6, 7.5x10^5,1.85x10^5, 4.7x10^4 copies/µl). In order to avoid template DNA loss, salmon DNA was used as a carrier for the low copy number range dilutions such that the total concentrations of nucleic acids in all dilutions are identical. qPCR was preformed with iCycler (BioRad) using Quantitect RT-PCR kit (Qiagen). The composition of the qPCR reactions are the same as RT-qPCR except the template was purified PCR product. Thermal cycle parameters were also identical to RT-qPCR so that results from DNA template can be compared with that from RNA template. All primer concentrations were set at 0.25 µM in all cDNA PCRs. For RT-qPCR analysis, two sets of primers respectively for chicken CD8a and RHACD were tested. The three levels of primer concentrations were 0.125, 0.25, and 0.5 µM. Total RNA was diluted to 80 ng/µl, 20 ng/µl, 5 ng/µl, 1.25 ng/µl, and 0.3125 ng/µl. Quadruplicated test was performed for each template concentration. Each well contained 20 µl in total including 2 µl of template. Thermal cycles were: reverse transcription at 50°C for 10 min, activation at 95°C for 15 min, 35 cycles of amplification (95°C for 15 s and 60°C for 60 s). Melting curve analysis was done for all reactions.

Three tests were conducted using CD8α DNA template. At high DNA copy number range , the efficiency of qPCR was 97%. The second and third qPCRs tests using low copy number range dilutions demonstrated efficiencies of 116% and 120% (see DNA diagram). With RNA as template, the efficiencies of RT-qPCR for CD8α primers at three concentrations 0.125, 0.25 and 0.5 µM were 97%, 95% and 97% respectively. Since these tests were performed separately, it is not possible to make strict comparisons between the efficiencies. However, these results were comparable to those with DNA template. Although the efficiencies were similar at the three primer concentration levels, higher primer concentrations showed consistently smaller Ct values for CD8a, suggest a slightly increased efficiencies.RHACD primer showed similar results. When primer concentration was 0.5 µM, the efficiency was 105%. When primer concentration was 0.25 µM, the efficiency was 99.7%. When primer concentration was 0.125 µM, the efficiency was 80%.

Higher levels of primer concentrations produce higher efficiencies. The RT-qPCR reagents must be very accurately mixed in order for the efficiency to be in an acceptable range. Sporadic high efficiencies may be avoided by eliminate pipetting errors. The results of qPCR with DNA template demonstrate that the linear relationship between the logarithmic of template concentration and threshold cycle hold at template concentration as low as 40,000 copies per reaction.

I learned about PCR’s though various sources:
Xiaofei Wang, PhD.Assistant Professor Endocrinology/Genomics Department of Biological Sciences Tennessee State University Nashville, TN37209 Tel: (615)963 2541

National Human Genome Ins