Notes: The full-length 3´ UTR of mouse RNA-binding protein HuR was amplified and cloned into psiCHECK™-1 Vector to create pEM429 plasmid. To generate radiolabeled RNA substrates for use in cleavage assays, RNA was synthesized from 1µg of linearized plasmid using 50µCi of [α-³²P]UTP, 0.8mM Ribo m⁷G Cap Analog and T7 RNA Polymerase by incubating for 1.5 hours at 37°C. The RNAs were then treated with 1 unit of RQ1 RNase-Free DNase per 1µg of template DNA for 15 minutes at 37°C before extracting with phenol:chloroform, precipitating with ethanol and resuspending in DEPC-treated water. The cleavage assay used 60fmol of ³²P-labeled substrate RNA with 10U Recombinant RNAsin Ribonuclease Inhibitor in a reaction incubated for 2.5 hours at 30°C. RNA probes were labeled with biotin using T7 or T3 RNA Polymerases with a biotin-UTP labeling NTP mixture and incubated for 2 hours at 37°C. The biotinylation reaction was then treated with RQ1 RNase-Free DNase following the same protocol used for radiolabeled RNA. To form HuR/RNA complexes, 2µg of biotinylated RNA was mixed with 100µg nuclear extract and 40 units Recombinant RNAsin^® Ribonuclease Inhibitor in a total volume of 20µl, and incubated for 30 minutes at room temperature. For CstF-64/RNA complexes, 1µg of biotinylated RNA was used and the complexes were stabilized by UV crosslinking using 10U Recombinant RNAsin Ribonuclease Inhibitor during the UV treatment. NIH 3T3 cells were UV crosslinked and either total or nuclear RNA used for immunoprecipitation. After extraction the RNAs were treated with RQ1 RNase-Free DNase for 15 minutes at 37°C before RT-PCR using HuR-specific primers. Total RNA purified from cultured cells were incubated with 50U/ml RQ1 RNase-Free DNase at 37°C for 30 minutes before use in RT-qPCR. (4187)

Notes: The authors studied hsp70 mRNA degradation in Drosophila Schneider cells. mRNA deadenylation and decay were monitored by Northern blot. Two of the Northern blot probes used to visualize the mRNA decay products were synthesized by transcription of linearized plasmids using T7 RNA Polymerase and [α-³²P] UTP. A population of deadenylated mRNA was created by hybridizing mRNA with oligo(dT) and treating with RNase H. CCR4•NOT was identified as the main deadenylase involved in mRNA decay, and the PAN2:PAN3 deadenylase was a minor contributor. RNA interference was used to knock down expression of PAN2 and CAF1, a subunit of CCR4•NOT, to assess the effect on mRNA decay. Reduced expression levels of PAN2 and CAF1 were confirmed by semi-quantitative RT-PCR and Western blotting, respectively. RT-PCR was performed using 1.5µg total RNA and 150 units of MMLV Reverse Transcriptase in a 25µl reaction. One microliter of the RT reaction was used as a template in an 80µl PCR using 0.5 units of GoTaq^® DNA Polymerase, 1.5mM MgCl₂ and 1X Green GoTaq^® Flexi Reaction Buffer. (3707)

Notes: T7 Polymerase and optimized buffer were used for in vitro transcription of various deletions in the open reading frame of RNA hypovirus CHV1-EP713. The  in vitro-transcribed deletion mutants and intact viral RNAs up to 12kb in size were then electroporated into Spheroplasts of C. parasitica and assessed based on their functionality.  (2728)

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: Transcribed cDNA for type X collagen using Promega's T7 RNA Polymerase. Translated this using Flexi^® Rabbit Reticulocyte Lysate System in presence of semi-permeabilized HT1080 cells to allow for correct assembly of fibrillar collagens. (0714)

Notes: RNase ONE™ Ribonuclease was used for RNase protection assays. The authors note that when they used a mixture of RNase A and T1 in place of RNase ONE™ Ribonuclease they had to be more diligent with their assays because the RNase A/T1 mixture was more likely to digest the protected fragment. RNase ONE™ Ribonuclease demonstrated single-stranded specificity in their experiments. (1492)

Notes: In this paper, Tfl DNA polymerase was used for two-step RT-PCR. The amplified fragments were cloned into the pGEM®-T Vector. Promega's Terminal Deoxynucleotidyl Transferase (TdT) was used to end label specific oligos for screening a frog brain cDNA library. T7 and T3 RNA polymerases were used to make digoxigenin labeled riboprobes for in situ hybridization. (1281)

Notes: The SP6 and T7 RNA Polymerases were used to produce RNA probes for Northern Analysis. The authors examined the half-life of the m4 muscarinic receptor in PC12 cells and transformed PC12 cells following treatment with either murine 2.5S NGF, human recombinant basic FGF or human recombinant EGF. PC12 cells were also treated with the three factors, nuclei isolated, and nuclear run-on transcription assay were performed. (0814)

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 Riboprobe^® in vitro Transcription System was used to produce RNA probes for Northern analysis. (0417)

Notes: T7 RNA Polymerase and SP6 RNA Polymerase were used to produce RNA probes labeled with digoxygenin-11-UTP. Transcripts were made in the presence of RNasin^® Ribonuclease Inhibitor in a standard transcription reaction. The ribonucleotides ATP, CTP and GTP were used at 1mM. Both UTP and digoxygenin-11-UTP were used at 0.5mM in the reaction. The RNA probe was used for in situ hybridization. (1563)

Notes: [³⁵S]labeled FR-19B (FGF-regulated 19B) protein was transcribed using the T7 RNA polymerase and translated in the TNT^® Coupled Reticulocyte Lysate System. A gel shift assay was performed with these proteins and double-stranded oligo from the SV40 enhancer. The results indicate that FR-19B specifically binds to the GT-IIC motif. (1804)