Single-strand cDNA was purified from aRNA through incubation with 0.5 µl of RNAseA (ThermoFisher Scientific) for 30 min at 37 °C. Finally, cDNA was amplified by PCR, which also attaches the Illumina small RNA barcodes and handles (Supplementary Table 3), by adding 25 µl...
One-step quantitative SYBR Green I RT-PCR was developed by amplifying cDNA template from viral RNA and usingin vitrotranscribed BVDV RNA to establish a standard curve. The assay had a detection limit as low as 100 copies/ml of BVDV RNA, a reaction efficiency of 103.2%, a correlation ...
Amplification of cDNA and primary molecular phylogeny based on β-actingene sequences of a freshwater crab,Sinopotamon yangtsekiense Amplification and sequence of the 尾-actin gene of Sinopotamon yangtsekiense were conducted with the reverse transcription polymerase chain reaction(RT-PCR... AB Gao,...
Optimization of procedures for real‐time PCR performance was carried out. Results. The transport medium, RNAlater, exhibited a high preservation effect against RNA degradation even after 8 days of storage at room temperature; one biopsy from each patient was sufficient for RNA extraction, cDNA ...
A total of 1 μg of RNA was reverse transcribed into cDNA and the genes of interests were amplified using the VeriQuest Fast SYBR Green qPCR (USB) with appropriate primers (Table S2). The PCR cycle conditions were as follows: 50 °C for 2 min., 95 °C for 5 min.,...
Specimen RNA was reverse-transcribed and co-amplified with a competitive template containing a gag sequence specific for BM5d that can be discriminated from that corresponding to wild-type cDNA by the presence of a unique restriction site, BglII . PCR products were quantified by means of ...
a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease...
All degenerate and quantita- tive RT-PCR primer pairs used for pure culture and environmental studies are outlined in Table 1. Sample cDNA was generated from mRNA transcripts using the DuraScript Enhanced Avian RT single- strand synthesis kit (Sigma Aldrich) according to the manufacturer's ...
The PCR primers and TaqMan probe targeted a 67-nucleotide (nt) sequence in the MoV M RNA segment. Using dilution series of a 849 nt RNA transcribed in vitro from cDNA clone pMoV4.1, the assay could detect down to a single MoV RNA equivalent, reliably detected 10 RNA copies and had a...
(San Diego, Calif.) following the isolation of RNA and reverse transcription, the obtained cDNA is spiked with a synthetic DNA molecule (competitor), which matches the targeted cDNA region in all positions, except a single base, and serves as an internal standard. The cDNA/competitor mixture ...