RT-PCR was carried out with total RNA from maize leaves using the indicated RT reaction temperatures. A 1.2 kb fragment of the GC-rich maize gl2 transcript was amplified. RT was carried out using the standard Omniscript RT protocol at the standard (37°C) and higher reaction temperatures as indicated.
RT-PCR was carried out with total RNA from maize leaves using the indicated RT reaction temperatures. A 1.2 kb fragment of the GC-rich maize gl2 transcript was amplified. RT was carried out using the standard MMLV RNase H- RT protocol (Supplier L) at the standard (42°C) and other reaction temperatures, as indicated. This standard protocol was carried out with the required preliminary denaturation step and the recommended RNase H digest step following RT.
The Omniscript RT Kit includes dNTPs and an optimized reaction buffer that, together with the high affinity of Omniscript Reverse Transcriptase for RNA, enables read-through of templates with high GC content or complex secondary structures. Due to this pre-optimization, tedious pipetting and pre-incubation steps are eliminated, and no additional RNase H digestion step is required.
Reverse transcription was carried out with different reverse transcriptases according to suppliers' specifications, using the indicated amounts of total RNA from HeLa cells. 1/20 of the reverse-transcription reaction was used in a 25-cycle PCR amplification with QIAGEN Taq DNA Polymerase. A 1.7 kb beta-actin fragment was amplified. Omniscript: Omniscript Reverse Transcriptase (QIAGEN); MMLV RNase H–: RNase H– reverse transcriptase from Moloney murine leukemia virus (Supplier L); MMLV: wild-type MMLV reverse transcriptase (Supplier L); AMV RNase H–: thermostable RNase H– reverse transcriptase from avian myeloblastosis virus (Supplier L); AMV: wild-type AMV reverse transcriptase (Supplier P).
A 700 nt in vitro transcript was reverse transcribed from 10 to 104 copies of starting template (in 200 ng total RNA), followed by PCR. 1/4 of each reverse-transcription reaction (corresponding to 2.5 to 2500 cDNA copies) was used for PCR amplification of a 330 bp fragment.