Abstract
Among the methods currently available for gene expression analysis, RT-PCR-based assays are the most suitable approaches for assessing mRNA expression levels in formalin-fixed and paraffin-embedded (FFPE) tissues because they allow the detection of target genes even when using relatively poor or degraded RNA. The reliability of gene expression results, however, can be affected by several technical sources of variability, most of which are referred to the reverse transcription step. This problem is further exacerbated when RNA from FFPE material is used, especially for the presence of inhibitory components and for the often unpredictable RNA degradation levels. In this chapter two alternative reverse transcription methods are described. They have been adapted to RNA from archival material and are useful when optimization of the assay is required because they are almost always based on home-made reagents. The protocols included in this chapter differ mainly for the nature of the reverse transcriptase and the priming strategy used. The choice of the most appropriate method depends especially on the amount of starting RNA and on the expected expression levels of the gene under investigation.
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- 1.
RNase H is an endonuclease that specifically degrades the RNA in RNA: DNA hybrids. It is commonly used to destroy the RNA template after cDNA synthesis, as well as in procedures such as nuclease protection assays. Since an RNase H activity is also present in the reverse transcriptases, they have been genetically altered to remove this activity, resulting in an increase of full-length cDNA products.
- 2.
DEPC is a carcinogen and should be handled with care under a fume hood.
- 3.
When testing mRNA sequences rich in secondary structures, the use of a thermostable reverse transcriptase such as Superscript III or AMVTM is recommended. In such case, the RT step should be carried out at 60°C if using amplicon-specific primer (see Sect. 19.3) or at lower temperatures if using oligo-dT or random primers.
- 4.
dUTP instead of dTTP may be used both in RT and in PCR reaction to avoid reamplification of carryover PCR products. In such case, an additional incubation of mixes at 50°C for 2′ with the enzyme uracil N-glycosilase is necessary before the transcription procedure. 10′ incubation at 95°C is performed to heat-inactivate the enzyme. Because UNG is not completely deactivated at 95°C, the PCR reaction temperatures should be kept higher than 55°C.
- 5.
The use of RNase inhibitor is critical for the successful processing of RNA as naked RNA from tissue samples is extremely susceptible to degradation by endogenous ribonucleases (RNases).
- 6.
Additional MgCl2 may be useful if the RNA has been previously digested by DNase according to the protocol described in the chapter about DNase digestion followed by heat inactivation, because the chelating properties of EDTA, used to inactivate DNase, can reduce free Mg2+ concentration
- 7.
Clean the pipettes with a disinfectant (e.g., Meliseptol®rapid) and leave them under the UV lamp for at least 10 min. Alternatively it is possible to autoclave the pipette depending on the provider instructions.
- 8.
Do not add more than 5 μg of total RNA per reaction because efficiency of cDNA synthesis can be reduced by higher RNA quantities. Use the same amount of RNA in all reactions.
- 9.
Total RNA is preferred to polyA RNA when reverse transcription is performed on RNA extracted from FFPE as polyA enrichment requires additional purification steps that cause RNA loss.
- 10.
When low amounts of RNA are obtained from microdissected or from very small FFPE samples, a fixed volume instead of a fixed quantity can be used for RT reaction.
- 11.
Refer to chapter dedicated to RNA extraction from FFPE for precautions against RNases contamination.
- 12.
DEPC is a carcinogen and should be handled with care under a fume hood.
- 13.
dUTP instead of dTTP may be used both in RT and in PCR reaction to avoid reamplification of carryover PCR products. In such case, an additional incubation of mixes at 50°C for 2′ with the enzyme uracil N-glycosilase is necessary before the transcription procedure. 10′ incubation at 95°C is performed to heat-inactivate the enzyme. Because UNG is not completely deactivated at 95°C, the PCR reaction temperatures should be kept higher than 55°C.
- 14.
As a general rule, primers should be between 15 and 25 bases long to maximize specificity, with a G/C content of around 50%. Avoid primers with secondary structures or with sequence complementarities at the 3′ ends that could form dimers. Specific software can be used for the design of primers for both endpoint and real-time PCR (e.g., Primer3, http://frodo.wi.mit.edu/primer3/, or IDTDNA, http://eu.idtdna.com/scitools/scitools.aspx).
- 15.
The use of RNase inhibitor is critical for the successful processing of RNA, as naked RNA from tissue samples is extremely susceptible to degradation by endogenous ribonucleases (RNases).
- 16.
Clean the pipettes with a disinfectant (e.g., Meliseptol®rapid) and leave them under the UV lamp for at least 10 min. Alternatively it is possible to autoclave the pipette depending on the provider instructions.
- 17.
Do not add more than 2 μg of total RNA per reaction because efficiency of cDNA synthesis can be reduced by higher RNA quantities. Use the same amount of RNA in all reactions.
- 18.
Total RNA is preferred to polyA RNA when reverse transcription is performed on RNA extracted from FFPE tissues as polyA enrichment requires additional purification steps that cause RNA loss and because polyA is degraded in these tissues.
- 19.
When low amounts of RNA are obtained from microdissected or from very small FFPE samples, a fixed volume instead of a fixed quantity can be used for the RT reaction.
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© 2011 Springer-Verlag Berlin Heidelberg
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Dotti, I., Bonin, S., Nardon, E. (2011). Reverse Transcription. In: Stanta, G. (eds) Guidelines for Molecular Analysis in Archive Tissues. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17890-0_19
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DOI: https://doi.org/10.1007/978-3-642-17890-0_19
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