RNasinRibonuclease Inhibitor (Promega)
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5 units of RNasin ribonuclease inhibitor (Promega), 10 U of Moloney murine leukemia virus reverse transcriptase (Promega), 10 mM Tris (pH 8.
5 [micro]M oligo(dT)18, 200 [micro]M dNTPs, 10 U RNAsin (Promega, Madison, WI, USA), 1xM-MLV RT buffer, and 300 U M-MLV reverse transcriptase (Promega, USA) in a volume of 50 [micro]l at 40[degrees]C for 1 h.
Throat swab and stool samples were suspended with phosphate-buffered saline (PBS) containing 10 U/mL RNasin (Promega, Madison, WI) and shaken for 10 min, followed by isolating supernatant by centrifuging as mentioned above.
For cDNA synthesis, 5 [micro]L of the extracted RNA was mixed with 1 [micro]mol of reverse primer V9207B (Table 3), 1x First Strand Buffer (Gibco BRL, Bethesda, MD), 1 mmol deoxynocleoside triphosphate (dNTPs), 80U RNAsin (Promega, Madison, WI), and 200 U SuperScript II Reverse Transcriptase (Gibco BRL, Bethesda, MD), and then incubated at 42[degrees]C for 1 h.
5 mmol/L dithiothreitol (Invitrogen), 500 [micro]mol/L deoxynucleotide triphosphates (Pharmacia), 50 [micro]mol/L random hexamer (Pharmacia), 1 unit/ [micro]L RNasin (Promega), and 10 U/[micro]L murine Moloney leukemia virus reverse transcriptase (Invitrogen).
The reaction was performed by PTC-200 programmable thermocycler (MJ research, USA) in a 20 [micro]l mixture consisting of 15 U avian myeloblastosis virus (AMV) reverse transcriptase, 20 U of RNasin ribonuclease inhibitor, 25 ng random primers, 5 mM MgCl2, 1 mM deoxynucleoside triphosphate (dNTP) mix and 1X reverse transcription buffer (10 mM Tris-HCl at pH 8.
2], 10 mM dithiothreitol (DTT), and 6 U RNasin in a total volume of 25 [micro]L.
The RNA was eluted in 30 [micro]L of Rnase-free water and 3 [micro]L of RNasin (Promega, Madison, WI) and used for RT-PCR.
We then added 1 [micro]l M-MLV reverse transcriptase, 5 [micro]l dNTP, 2 [micro]l RNasin, and 5 [micro]l 5.
5 mM dNTP mix, and 15 ng/[micro]L random primers, 1x first-strand buffer, 10 mM dithiothreitol, 27 U Rnasin RNase inhibitor (Promega, Madison, WI), and 800 U superscript reverse transcriptase (Invitrogen).