biologist's perspective
sorry to embarass you, but i fucking hate know-it-alls
the series of reactions goes like this;
total RNA extraction (between 5 and 20 ug); it has gotta be clean, suggest QIAGEN RNAEasy Kit
1st strand cDNA synthesis:
use poly T oligo to prime the polymerization, do a 70C annealing step for 10 minutes, add the 1st strand buffer DTT, dNTP's, 5 min. at 45C, add reverse transcriptase, incubate 45C 1 hour.
2nd strand synthesis
add
buffer, dNTP's, ligase, DNA Pol1, RNaseH, incubate 4 hrs. add T4 DNA polymerase. incubate 5 minutes. add EDTA
To incorporate label, IVT reaction (in vitro transcription) this turns the cDNA into cRNA. we use an ENZO IVT kit which includes the enzyme, RNase inhibitor, ribonucleotides, buffer and DTT.
this is used for the hybridization after we add spike controls.
you DumbAss!