Total RNA, prepared as described above, was treated with RNases A/T1 (Thermo Fisher Scientific) with concentrations of 0, 1.5, and 15 units at 37° for 15 min. The treated RNA was precipitated and reverse transcribed into cDNA by SuperScript III Reverse Transcriptase (Life Technologies) primed with random primers (Thermo Fisher Scientific) as the template for PCR. A seminested PCR was used to amplify regions inside the expected dsRNA (primers ac12 + ac6 followed by ac4 + ac6) to detect dsRNA. Genomic DNA contamination was not detected by using primer ac13 + ac14.
Rnases a t1
Manufactured by Thermo Fisher Scientific
RNases A/T1 are purified ribonuclease enzymes that degrade single-stranded RNA. RNase A is derived from bovine pancreas, while RNase T1 is derived from Aspergillus oryzae. These enzymes are commonly used in molecular biology applications to remove unwanted RNA from samples.
Automatically generated - may contain errors
2 protocols using rnases a t1
1
dsRNA detection via RNase and RT-PCR
2
RNA Extraction and cDNA Synthesis
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Total RNA, treated with DNase I (NEB), prepared as described above was treated with RNasesA/T1 (Thermo Scientific) with concentrations of 0 U, 1.5 U and 15 U at 37 o C for 15 min. The treated RNA was precipitated and reverse transcribed into cDNA by SuperScript® III Reverse Transcriptase (Life Technologies) primed with random primers (Thermo (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted January 8, 2020. ; https://doi.org/10.1101/2020.01.07.897926 doi: bioRxiv preprint Scientific) as the template for PCR. First round of PCR used primers a12 + a6, followed with the nested PCR using primers a4 + a6.
The copyright holder for this preprint this version posted January 8, 2020. ; https://doi.org/10.1101/2020.01.07.897926 doi: bioRxiv preprint Scientific) as the template for PCR. First round of PCR used primers a12 + a6, followed with the nested PCR using primers a4 + a6.
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