Editing Reverse transcription polymerase chain reaction (section)

== Challenges ==

Despite its major advantages, RT-PCR is not without drawbacks. The exponential growth of the reverse transcribed [[complementary DNA]] (cDNA) during the multiple cycles of PCR produces inaccurate end point quantification due to the difficulty in maintaining linearity.<ref name="pmid14664723">{{cite journal |author=Shiao YH |title=A new reverse transcription-polymerase chain reaction method for accurate quantification |journal=BMC Biotechnol. |volume=3 |page=22 |date=December 2003 |pmid=14664723 |pmc=317330 |doi=10.1186/1472-6750-3-22 |doi-access=free }}</ref> In order to provide accurate detection and quantification of RNA content in a sample, qRT-PCR was developed using fluorescence-based modification to monitor the amplification products during each cycle of PCR. The extreme sensitivity of the technique can be a double-edged sword since even the slightest DNA contamination can lead to undesirable results.<ref name="pmid9464395">{{cite journal |vauthors=Gettemy JM, Ma B, Alic M, Gold MH |title=Reverse transcription-PCR analysis of the regulation of the manganese peroxidase gene family |journal=Appl. Environ. Microbiol. |volume=64 |issue=2 |pages=569–74 |date=February 1998 |pmid=9464395 |pmc=106084 |doi= 10.1128/AEM.64.2.569-574.1998|bibcode=1998ApEnM..64..569G }}</ref> A simple method for elimination of false positive results is to include anchors, or [[Webtag (Software)|tags]], to the 5' region of a gene specific primer.<ref>{{cite journal|title=A simple method for elimination of false positive results in RT-PCR|journal=J Biochem Mol Biol|date=2002-03-31|first=Fatima|last=Martel|author2=Dirk Grundemann |author3=Edgar Schöig |volume=35|issue=2|pages=248–250|pmid=12297038|doi=10.5483/BMBRep.2002.35.2.248 |doi-access=free}}</ref> Additionally, planning and design of quantification studies can be technically challenging due to the existence of numerous sources of variation including template concentration and amplification efficiency.<ref name="pmid12618301">{{cite journal |vauthors=Ramakers C, Ruijter JM, Deprez RH, Moorman AF |title=Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data |journal=Neurosci. Lett. |volume=339 |issue=1 |pages=62–6 |date=March 2003 |pmid=12618301 |doi= 10.1016/S0304-3940(02)01423-4|s2cid=9459695 }}</ref> Spiking in a known quantity of RNA into a sample, adding a series of RNA dilutions generating a standard curve, and adding in a no template copy sample (no cDNA) may used as controls.