Reverse Transcription Real-Time PCR Protocol for Gene Expression Analyses

Abstract

Gene expression differences between healthy and atherosclerotic arteries are important to understand cellular processes involved in the progression and development of atherosclerotic disease. This protocol describes the experimental procedure for real-time reverse transcription PCR.

Appendices

Appendices

1.1 Appendix 1

1.1.1 Picking Random Primers and Oligo dT

Oligo dT primers are a favorite choice for two-step cDNA synthesis reactions because of their specificity for mRNA and because they allow many different targets to be studied from the same cDNA pool. However, because they always initiate reverse transcription at the 3′ end of the transcript, difficult secondary structure may lead to incomplete cDNA synthesis. Oligo dT priming of fragmented RNA, such as that isolated from Formalin-Fixed Paraffin-Embedded (FFPE) samples, may also be problematic. Nonetheless, as long as the primers are designed near the 3′ end of the target, premature termination downstream of this location is less of an issue.

Random primers are useful for synthesizing large pools of cDNA. They are also ideal for non-polyadenylated RNA, such as bacterial RNA, because they anneal throughout the target molecule. Degraded transcripts such as FFPE samples and secondary structure within the RNA do not pose as big a problem with random primers as they do with gene-specific primers and oligo dT primers. While increased cDNA yield is a benefit, data has shown that random primers can overestimate copy number when used in real-time RT-PCR experiments. Employing a combination of random and oligo dT primers can sometimes increase data quality by combining the benefits of both if used in the same first-strand cDNA synthesis reaction. Random primers are used only in two-step qRT-PCR reactions.

1.2 Appendix 2

1.2.1 Picking Housekeeping Gene

Relative quantification is a powerful technique that is commonly used to study RNA gene expression. In relative quantification the expression of a target gene is measured with respect to a stably expressed reference gene (so-called housekeeping gene); the two gene levels are expressed as a ratio.

Housekeeping genes must meet certain criteria before they can be effective reference genes. Housekeeping genes encode proteins that are essential for maintenance of cell function. For instance, housekeeping genes which code for components of the cytoskeleton (e.g., beta-actin, alpha-tubulin), components of the major histocompatibility complex (such as beta-2-microglobulin), enzymes of the glycolytic pathway (GAPDH (glyceraldehyde-3-phosphate dehydrogenase)) or ribosomal subunits appear to be expressed ubiquitously. However, several reports indicate that the expression of housekeeping genes is actively regulated; levels may vary across tissues and different types of cells, during cell proliferation and stages of development, or due to experimental treatment of cells.

Therefore, when choosing a housekeeping gene as a reference for relative quantification, one must identify a gene whose expression level remains relatively constant for a certain experimental setup. In fact, it is usually necessary to test a panel of housekeeping genes experimentally to find one that is not regulated in the investigated system. Since choosing an appropriate reference gene is critical for accurate quantitative RNA analysis, the behavior of candidate genes in different cell types and cell metabolic stages should be carefully examined.

1.3 Appendix 3

1.3.1 No-Template Control (NTC)

Controls in real-time PCR reactions prove that signal obtained from experimental samples represent the amplicon of interest, thereby validating specificity. All experiments should include a no-template control (NTC), and qRT-PCR reactions should also include a no-reverse transcriptase control (no-RT). NTC controls should contain all reaction components except the cDNA sample. Amplification detected in these wells is due to either primer-dimers or contamination with completed PCR reaction product. This type of contamination can make expression levels look higher than they actually are. No-RT reactions should contain all reaction components except the reverse transcriptase. If amplification products are seen in no-RT control reactions, this indicates that DNA was amplified rather than cDNA. This can also artificially inflate apparent expression levels in experimental samples

1.4 Appendix 4

1.4.1 Troubleshooting (Listed Some Major Causes for Real-Time PCR Failures)

Little or no PCR product: Poor quality of PCR templates, primers, or reagents may lead to PCR failures. First, please include appropriate PCR controls to eliminate these possibilities. Some genes are expressed transiently or only in certain tissues. In our experience, this is the most likely cause for negative PCR results. Please read literature for the gene expression patterns. One caveat is that microarrays are not always reliable at measuring gene expressions. After switching to the appropriate templates, we obtained positive PCR results in contrast to the otherwise negative PCRs (see our paper for more details).

Poor PCR amplification efficiency: The accuracy of real-time PCR is highly dependent on PCR efficiency. A reasonable efficiency should be at least 80 %. Poor primer quality is the leading cause for poor PCR efficiency. In this case, the PCR amplification curve usually reaches plateau early and the final fluorescence intensity is significantly lower than that of most other PCRs. This problem may be solved with re-synthesized primers.

Primer dimmer: Primer dimer may be occasionally observed if the gene expression level is very low. If this is the case, increasing the template amount may help eliminate the primer dimer formation.

Multiple bands on gel or multiple peaks in the melting curve: Agarose gel electrophoresis or melting curve analysis may not always reliably measure PCR specificity. From our experience, bimodal melting curves are sometimes observed for long amplicons (>200 bp) even when the PCRs are specific. The observed heterogeneity in melting temperature is due to internal sequence inhomogeneity (e.g. independently melting blocks of high and low GC content) rather than non-specific amplicon. On the other hand, for short amplicons (<150 bp) very weak (and fussy) bands migrating ahead of the major specific bands are sometimes observed on agarose gel. These weak bands are super-structured or single-stranded version of the specific amplicons in equilibrium state and therefore should be considered specific. Although gel electrophoresis or melting curve analysis alone may not be 100 % reliable, the combination of both can always reveal PCR specificity in our experience.

Non-specific amplicons: Non-specific amplicons, identified by both gel electrophoresis and melting curve analysis, give misleading real-time PCR result. To avoid this problem, please make sure to perform hot-start PCR and use at least 60 °C annealing temperature. We noticed not all hot-start Taq polymerases are equally efficient at suppressing polymerase activity during sample setup. The SYBR Green PCR master mix described here always gives us satisfactory results. If the non-specific amplicon is persistent, you have to choose a different primer pair for the gene of interest.