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RT reaction mix was prepared, containing 5 [micro]l 5x Moloney murine leukemia virus reverse transcriptase (MMLV) reaction buffer, 3.4 [micro]l 10 mM dNTPs, 0.6 [micro]l RNasin and 1 [micro]l MMLV reverse transcriptase, and 15 [micro]l of random primer/RNA mix and reconstituted to 25 [micro]l with DNase-free water (all Promega) and performed in a thermocycler at 37[degrees]C for 1 h.
Ten L of reverse transcriptase buffer (5X) 2 unit of MMLV-RT (Fermentas) 2 unit of RNAse inhibitor (RNAsin Fermentas) 0.6 L of dNTPs (25 mM) and 20 L of sterile water were added and the mix was incubated at 42C for 60 min.
Reverse transcription reactions were performed as follows: 2 [micro]L total RNA (1 pg/[micro]L), 4 [micro]L 5 x buffer, 2 [micro]L Oligo dT, 1 [micro]L, RNasin (40 U/[micro]L), 1 [micro]L dNTP mix, 1 [micro]L reverse transcriptase (200 U/[micro]L).
The reverse transcription reaction was performed in a final volume of 20.55 [micro]l containing 2 [micro]l of RNA template, 5 [micro]l of random primer N6 (100 [micro]M), 10 [micro]l of 2 x reverse transcription buffer, 3 [micro]l of dNTP (2.5 mM), 0.4 [micro]l of MMLV Rtase (200U/[micro]l), and 0.15 [micro]l of Rnasin (40U/[micro]l).
For each reaction 1 mg of RNA, 5 mM oligo([dT.sub.12]) primer, 20 mM dNTP, 40 mU Rnasin and 300 U AMV reverse transcriptase and water up to 20 mL final volume were mixed and incubated at room temperature for 15 min.
The cDNA was directly synthesized in the same microplate by adding dNTPs (final concentration, 5 mmol/L), Moloney murine leukemia virus reverse transcriptase (final concentration, 2.7 U/[micro]L), and RNasin (0.13 U/[micro]L) (Invitrogen) and incubated at 37[degrees]C for 2 h.
Two [micro]L DNA digestion solution was added to a cocktail mixture containing 1 [micro]L dNTP, 1 [micro]L 50 mM 3' anchor primer containing [57CT CTAAGCTT(T) n-37 ], 2 [micro]L Mg[Cl.sub.2],1 [micro]L 10x buffer, 0.25 [micro]L RNasin, 0.25 [micro]L AMV reverse transcriptase, and 4.5 [micro]L sterile dd[H.sub.2]O (Promega, USA).
One pg of RNA was denatured at 65[degrees]C for 5 min and reverse transcribed in a 20 [micro]L reaction mixture containing 4 [micro]L of 5x reverse-transcription (RT) buffer (Invitrogen), 0.2 [micro]mol of DTT, 100 U of Superscript II (Invitrogen), 20 U of RNasin (Promega, Madison, WI), 10 nmol of each dNTP, and 100pmol of random hexamers.
The reverse transcription step was performed with 25 [micro]L of the viral RNA and 15 [micro]L of a reaction mixture containing 200 [micro]M deoxynucleoside triphosphates (dNTPs) (GE Healthcare, Austria), 5mM dithiothreitol (DTT) (Invitrogen, Germany), 0.4 mM random primer (Invitrogen, Germany), 20-unit RNasin RNase inhibitor (Promega, USA), 100-unit Moloney murine leukemia virus reverse transcriptase enzyme (M-MLV) (Invitrogen, Germany), and a reaction buffer (Invitrogen, Germany).
TRI-Reagent and RNA Secure Reagent were purchased from Ambion (USA); the cDNA synthesis MMLV RT kit and PCR kit qPCRmix-HS were from Evrogen (Russia); RNasin and RQ1 DNase were from Promega (USA); oligonucleotides (primers) for analysis of the rat genes Cyp1a1, Cyp1a2, Cyp1b1, Gsta1, Nqo1, Aldh3a1, Ugt1a6, Ugt1a9, AhR, Nrf2, Gsr, Txnrd1, Hmox1, and Gapdh were from Syntol (Russia).
The PCR mixture was prepared in 25 [micro]L reaction volume containing 10 mM each of dNTPs mix, 0.2 mM Tfl DNA polymerase (5U/[micro]L), 0.1 U AMV (5U/[micro]L), 0.1 U recombinant RNasin ribonuclease inhibitor (400 U/[micro]L), 0.8 U MgS[O.sub.4] (25 mM), 20 pmol of each of the forward and reverse primer, 5.0 [micro]L of 1 times buffer, and 1 [micro]L RNA or DNA template.
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- RNase 5
- RNase 5
- RNase 5
- RNase 5
- RNase A
- RNase alpha
- RNase D
- RNase D domain
- RNase H
- RNase I
- RNase II
- RNase III
- RNase P
- RNase protection assay
- RNase T1
- RNase T2
- RNase U2
- RNase U4
- RNAV approach
- RNAV Approach Focus Group
- RNAV waypoint