Reverse Transcription of RNA- 20l rxn
4l RT buffer
9l H2O (RNA to be included in this vol but added last)
2l 0.1M DTT
1l oligo dT primer
2l 10mM dNTP's
1l Reverse Transcriptase
incubate for 1hr at 37C then heat inactivate at 70C for 10mins.
All reagents should be diluted in dH20 that has been dep'd and autoclaved.
Add all reagents except the RT and RNA and let them sit in the presence of the RNase for 15 mins.
I use 1.5 ug of Poly A+ RNA in this rxn. This is the quivalent of ~ 25-50 ug total RNA. (X-H uses much less).
I have seen consistently better results with oligo dT primed RNA even for very long RNA where 5 prime regions are to be amplified.
Many people swear by superscript in our experience biolabs is just as good they are equivalent in units but the companies count them differently. Superscript can be used at higher temperature if secodary structure is a problem.
I have put between 0.01 - 5.0l of this rxn into a standard 50l PCR rxn.
0.01l is the 1st strand derived from 0.00075 ug of Poly A+ RNA which is the equivalent of 0.15ug of total RNA (X-H's method works out to use the eqivalent of 0.12ug of total RNA in the RT-PCR)
Controls should include:
1) primers alone
2)cDNA +ve control
3) first strand without RT to test for genomic contamination- we have found this to be a problem - so if possible choose primers from either side of an intron.
DC-DX and DC-BAX oligos work well as + controls of bovine RNA using 23a cDNA as PCR control template. Expect a 1.3 kb fragment
PG 2124F and 2357R oligos work well as + controls of bovine RNA
BC-NF and 1140R oligos work well as + controls of bovine RNA
Dilute cDNAs 1/100