Notes: A novel cell fusing agent virus (CFAV) strain was identified in mosquito populations in northern Brazil, shedding light on possible surveillance methods for other flaviviruses such as Zika virus. ProNex® size-selective beads were used alongside Promega AMV Reverse Transcriptase and DNA Polymerase I Large Fragment in the next generation sequencing workflow. (5217)

Notes: In this report, the original investigators who found XMRV and pMLV (polytropic murine leukemia virus) in blood of subjects with Chronic Fatigue Syndrome (CFS) report that this association is not confirmed in a blinded analysis of samples from rigorously characterized subjects.

The CDC performed nucleic acid testing assays. Plasma was centrifuged and RNA isolated from the pellet. Quantitative real-time RT-PCR assays (qRT-PCR) for generic pMLV/XMRV pro (protease) and gag detection were performed on RNA extracts, using the AccessQuick™ RT-PCR System and an AgPath one-step RT-PCR kit.

ArrayScript RT and AmpliTaq Gold DNA polymerase were used for cDNA synthesis and amplification in the pro and gag qRT-PCR assays, respectively. A third PCR was done using the primers XPOLOF and XPOLOR, followed by a nested PCR with the primers XPOLIF and XPOLIR for the generic detection of MLV/XMRV 216-bp pol sequences. For this reaction, cDNA synthesis and amplification of RNA was done using Promega AMV Reverse Transcriptase and a RobustI RT-PCR kit. Each PCR experiment included 20 water-only reactions to control for contamination. (4300)

Notes: This paper describes a method for detection of polyA RNA in permeablized cells and cell sections. The method is based on incorporation of 5-bromo-2´-deoxyuridine (BrdUTP) into cDNA by AMV reverse transcriptase. The BrdUTP was easily detectable in DNA-RNA duplexes, and undetectable in DNA-DNA duplexes, making it possible to use the method to detect RNA in situ. RNasin® Ribonuclease Inhibitor was used in the reverse transcriptase reaction mix. (4226)

Notes: To study how chemotherapy affects hair follicles (HF), the researchers microdissected and cultured human anagen VI scalp follicles and treated the cells with 4-Hydroperoxycyclophosphamide (4-HC) at similar concentrations to those received during therapy. After incubation for 24 hours with 30µmol/L of 4-HC, the HFs were homogenized and genomic DNA was purified using the Wizard^® SV Genomic DNA Purification System. To determine if the treatment induced the mitochondrial common DNA deletion, the isolated DNA was subjected to real-time PCR. Further examination of gene expression was performed by isolating total RNA from two HF samples, reverse transcribing 3µg of the RNA using 15U of AMV Reverse Transcriptase and 0.025 µg/µl random primers, and amplifying seven human genes in a TaqMan^® Assay. (3746)

Notes: Promega reverse transcription buffer, AMV reverse transcriptase, and RNAsin^® RNAse inhibitor were used in reverse transcription reactions of total RNA from HepG2 heptatoma cells. PCR products were cloned into the pGEMT vector. (2613)

Notes: The RNAgents^® Total RNA Isolation System was used to isolate total RNA from 100ml cultures of S. enterica grown to an OD₆₀₀ of 1.0 in permissive conditions of limited oxygen and high osmolarity. The RNA was used in primer extension analysis with AMV Reverse Transcriptase. (2574)

Notes: The authors investigated expression of BRCA1 from alternative transcripts possessing different 5′ UTRs. RT-PCR using Promega's AMV reverse transcriptase was performed to generate cDNAs from total RNA isolated from human tissue. Promega's Taq DNA Polymerase was used for the PCR reaction. The authors also prepared two mRNAs that contained one or the other of the 2 alternative 5′ UTRs (ex1a or ex1b) fused with the luciferase coding region. To generate the corresponding DNA for these constructs, the luciferase coding region was amplified from Promega's pGEM® Vector and ligated to PCR-amplified ex1a or exlb. These ligation products served as the template amplifying two cDNA constructs (exla-luc or ex1b-luc). Ten additional cDNAs were made containing a variety of changes to the 5′UTR region of BRCA1. In vitro translation experiments were performed to determine how the composition of the 5′UTR affected protein expression levels (using Promega's RNasin to protect transcribed mRNA; T7 polymerase buffer; and rabbit reticulocyte and wheat germ extracts for translation). The authors conclude that secondary structures associated with elements in the longer 5′UTR reduced translation rates and could be responsible for the reduced expression of BRCA1 often associated with spontaneous ovarian and breast cancers. (2441)

Notes: The study of a carbapenem resistant strain of Klebsiella pneumoniae lead to the description of a novel form of β-lactamase, which confers resistance to imipenem, meropenem, extended-spectrum cepahlosporins, and aztreonam. Total RNA used in a primer extension assay was isolated with the SV Total RNA Isolation System . Primer extension reactions were performed with Promega's Primer Extension System- AMV Reverse Transcriptase to determine the transcriptional start site of the KPC-1 β-lactamase gene. The KPC-1 β-lactamase gene was then sequenced with the fmol® DNA Sequencing System (2300)

Notes: Total RNA was isolated from Alteromonas sp. strain O-7 for Northern blot analysis of the aprII gene to show that aprII is upregulated under iron rich growth conditions. The transcriptional start site of this gene was mapped in a primer extension reaction using Promega's AMV reverse transcriptase. (2304)

Notes: The SV Total RNA Isolation System was used to isolate total RNA from rat endothelial cells. The isolated RNA was used for RT-PCR and semi-quantitative RT-PCR with cDNA generated with AMV Reverse Transcriptase. (2175)

Notes: Poly A+ RNA was isolated from the total RNA of three different strains of Saccharomyces cerevisiae, wild-type (JC103), CS165 and JC408, using the PolyATtract^® mRNA Isolation System. The isolated polyA+ RNA was used to make biotinylated cRNAs via a referenced method using AMV Reverse Transcriptase. The resulting probes were used in microarray analysis. (2696)

Notes: Total RNA was isolated from Rhizobium leguminosarum bv. trifolii using the SV Total RNA Isolation System. This RNA was used in a primer extension assay using AMV Reverse Transcriptase to determine the transcriptional start site of the mat operon. Nested deletions during the sequencing of the mat promoter were prepared. The firefly luciferase gene from pGL3 Basic was used to construct a reporter construct containing the mat promoter to study transcriptional effects of MatR. The interaction of the MatR protein with the mat operator was mapped. (2308)

Notes: Total RNA was isolated from 3-day-old mung bean hypocotyls using the SV Total RNA Isolation System. RT for RT-PCR was performed with the Reverse Transcription System. (0825)

Notes: Luciferase reporter constructs were cotransfected into 3T3-L1 preadipocytes with the pRL-CMV Vector at a 25:1 ratio and luciferase activities were determined with the Dual-Luciferase^® Reporter Assay System. RT-PCR was accomplished with AMV Reverse Transcriptase and Taq DNA Polymerase. RNase Protection Assay were performed with the aid of the RNase ONE™ Ribonuclease. (0988)

Notes: For the RT-PCR experiment, total RNA isolated from fly heads was reverse transcribed by Promega AMV-RT and then the cDNA was amplified by Promega Taq DNA polymerase. (0574)

Notes: First-strand cDNA was synthesized by use of the Reverse Transcription System and random primers. (0728)

Notes: PCR products were excised, eluted, and precipitated prior to sequencing with the fmol^® DNA Cycle Sequencing System. Promega's RNasin^® Ribonuclease Inhibitor and AMV Reverse Transcriptase were used in a reverse transcription assay. (2065)

Notes: The authors used the PolyATtract® mRNA Isolation System to isolate mRNA from total RNA. They used this mRNA-rich fraction for primer extension analysis using AMV Reverse Transcriptase. The Wizard® Plus Midipreps DNA Purification System was used for various plasmid isolations. The Dual-Luciferase® Reporter Assay System was used to study promoters cloned into the pGL3-Basic Vector. The pRL-SV40 was used as a transfection control plasmid. (0095)

Notes: The RNasin Ribonuclease Inhibitor was used to protect RNA transcripts during primer extension analysis and in vitro transcription of RNA probes. The resultant RNA probes were used for RNase protection assays. (1639)

Notes: The authors used Promega's AMV Primer Extension System and Access RT-PCR System for this study. (2216)

Notes: The TNT^® Coupled Transcription/Translation System was used to verify the 25.3kDa size of the cloned receptor. AMV reverse transcriptase was used for first strand cDNA synthesis prior to PCR. (1520)

Notes: Promega's AMV Reverse Transcriptase and Taq DNA Polymerase were used to perform RT-PCR in this study. (2021)

Notes: Poly(A)+ RNA was isolated from RIN rat insulinoma cell total RNA with PolyATtract® mRNA Isolation System III. (1264)

Notes: Poly(A+) RNA was isolated from RIN rat insulinoma cell total RNA and was used for Northern blots. The authors used Promega's PolyATtract^® mRNA Isolation System and AMV Reverse Transcriptase in this study. (2107)

Notes: Poly(A)+ RNA was isolated from mouse brain total RNA and used for Northern analysis. The authors used Promega's PolyATtract^® mRNA Isolation System, AMV Reverse Transcriptase and RNasin^® Ribonuclease Inhibitor in this study. (2104)