Phenol Free Rna Binding Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids"
Article Title: Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids
Figure Legend Snippet: Nonvesicular tRNA halves circulating in human biofluids and intracellular glycine tiRNAs are predominantly nicked tRNAs. A) Purified RNA from arsenite-treated U2-OS cells was separated on a Superdex 75 column using an FPLC system. Inset: northern blot of intracellular RNAs showing the presence of tRNA halves in the input. Selected fractions from (A) were analyzed by northern blot (B) or by stem-loop RT-qPCR (C). C q values were normalized to the fraction containing the highest signal. A-C bottom panels: the RNA was heat-denatured before injection. D) Cells were transfected with synthetic 5’ tRNA Gly [9 GG/AA ], then lysed with SDS as described in ( Tosar et al., 2018 ). The lysate was separated by SEC and fractions analyzed by SL-RT-qPCR using primers specific for the 9 GG/AA sequence. E–F) Separation by SEC of purified RNA from Proteinase K-treated ultracentrifugation supernatants of human serum (E) or CSF (F). Selected eluted fractions were analyzed by SL-RT-qPCR using primers specific for 5’ tRNA Gly GCC halves of 30 nt (red) and miR-21-5p (green). For reference, a tRNA icon in this figure indicates fractions where full-length tRNAs are expected to elute (if present).
Techniques Used: Purification, Fast Protein Liquid Chromatography, Northern Blot, Quantitative RT-PCR, Injection, Transfection, Sequencing
Figure Legend Snippet: Identification of stable nonvesicular RNAs. Northern blot of different rRNAs and tRNAs after incubating purified RNA (A) or ribosomes (B) from human cells in 10% FBS. C-D) Northern blot of 5’ tRNA Gly GCC (D, left) or 5’ 28S rRNA-derived fragments (D, right) in extracellular nonvesicular fractions purified by density gradients (C). U2-OS cells were treated or not with NaAsO 2 (ARS) before collecting the cell-conditioned medium (CCM). E) Read coverage in small RNA-seq data of extracellular ribosomes (from Tosar et al., 2020 ), revealing enrichment of 40 nt 5’-derived small RNAs among all other 28S rRNA-derived fragments.
Techniques Used: Northern Blot, Purification, Derivative Assay, RNA Sequencing Assay
Figure Legend Snippet: Nicked tRNAs protect tRNA halves from degradation, are dissociated by phenol, and can be repaired by RtcB. RNA purification by the miRNeasy micro kit (A) or TRIzol (TRI, B) impairs enzymatic (PNK+Rnl1) nicked tRNA repair (blue arrows). C) Purification of RNase1-treated RNA by SPE and re-exposure to RNase 1, with or without previous heat denaturation. D) Northern blot of RNAse1-treated RNA purified by SPE, TRIzol, miRNeasy or heated before SPE purification, after separation in native gels. E) Nicked tRNA repair with RtCB from E. coli .
Techniques Used: Purification, Northern Blot
Figure Legend Snippet: Most tRNA halves identified by northern blot are nicked tRNAs. A) RNase 1-treated RNA (60 min) was incubated with the indicated enzymatic combinations. Repair of tRNA Gly GCC was analyzed by northern blot using a 5’-targeting probe. B) schematic representation of the nicked tRNA repair strategy and 5’ (blue) and 3’ (orange) probe binding sites in tRNA Gly GCC . C) Design of a third probe targeting the anticodon loop (ACL) of tRNA Gly GCC and tRNA Asp GUC . D) enzymatic repair of tRNA Gly GCC evidenced with either the 5’, ACL or 3’ probes. NT: fragmented RNA purified by SPE but without treatment with the enzymatic repair cocktail. (-)PNK: mutant PNK lacking phosphatase activity. Δ: heat.
Techniques Used: Northern Blot, Incubation, Binding Assay, Purification, Mutagenesis, Activity Assay
Figure Legend Snippet: Naked tRNA halves are extremely stable in human biofluids. Northern blot of several noncoding transcripts after incubating purified RNA from human cells in 10% FBS or with recombinant human RNase 1 for different periods (A). Samples were also incubated in human urine, 10% serum and CSF (B). Half-lives in CSF were calculated for all tested RNAs and shown in (C). r-RNase1: recombinant human RNase 1.
Techniques Used: Northern Blot, Purification, Recombinant, Incubation
2) Product Images from "Aging-Associated Differences in Epitranscriptomic m6A Regulation in Response to Acute Cardiac Ischemia/Reperfusion Injury in Female Mice"
Article Title: Aging-Associated Differences in Epitranscriptomic m6A Regulation in Response to Acute Cardiac Ischemia/Reperfusion Injury in Female Mice
Journal: Frontiers in Pharmacology
Figure Legend Snippet: In vitro hypoxia/reperfusion (iH/R) stress regulates m6A levels of key anti-apoptotic and pro-apoptotic genes. ( A–C ). Ctrl and Mettl3 mofified HL1 cardiomyocytes were subjected to sham or iH/R, and total RNA was subjected to m6A RIP, followed by RT-qPCR using primers for the indicated pro-apoptotic genes (Bax and PTEN) and anti-apoptitic gene (Bcl2). Values are normalized to input. n = 3–5, * p
Techniques Used: In Vitro, Quantitative RT-PCR
3) Product Images from "Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication"
Article Title: Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication
Journal: Journal of Virology
Figure Legend Snippet: RNA binding of cleavage-resistant PCBP2. Radiolabeled poliovirus stem-loop RNA was used in mobility shift assays to determine the ability of the cleavage-resistant PCBP2 to bind to stem-loop I or IV derived from the 5′ NCR. (A) Radiolabeled poliovirus stem-loop IV (SL-IV) was incubated with increasing amounts of PCBP2 (lanes 2 to 4) or uncleavable PCBP2 (QS→ID) (lanes 5 to 7). (B) Radiolabeled poliovirus stem-loop I (SL-I) was incubated with poliovirus 3CD alone (lane 2) or with PCBP2 alone (lanes 3 and 4) or PCBP2 (QS→ID) alone (lanes 7 and 8). To analyze ternary complex formation, increasing amounts of poliovirus 3CD were added to wild-type (lanes 5 and 6) or uncleavable (lanes 9 and 10) PCBP2. The arrows to the right of the gel image indicate probe alone (Free Probe), RNP complex formation, and ternary complex formation.
Techniques Used: RNA Binding Assay, Mobility Shift, Derivative Assay, Incubation
Figure Legend Snippet: Functional analysis of uncleavable PCBP2 (QS→ID) during poliovirus translation. Poliovirus virion RNA was translated in vitro in PCBP-depleted HeLa cytoplasmic extracts in the presence of [ 35 S]methionine. Poliovirus proteins were resolved by SDS-PAGE and detected by autoradiography. Translation reactions were carried out in mock-depleted (lane 1) or PCBP-depleted (lanes 2 to 6) HeLa cytoplasmic extracts. No added recombinant protein (lane 2), 100 nM or 500 nM recombinant wild-type PCBP2 (lanes 3 and 4), and 100 nM or 500 nM purified recombinant PCBP2 (QS→ID) (lanes 5 and 6) were analyzed for rescue of viral translation levels. Viral proteins are indicated to the left of the gel. WT, wild type.
Techniques Used: Functional Assay, In Vitro, SDS Page, Autoradiography, Recombinant, Purification
Figure Legend Snippet: In vitro translation of poliovirus RNA in the presence of wild-type or uncleavable PCBP2. HeLa cytoplasmic extract was preincubated with poly(rC) RNA to bind and sequester PCBP2. Poliovirus virion RNA was then added with or without increasing amounts of recombinant wild-type PCBP2 or uncleavable PCBP2 (QS→ID). (A) Translation of [ 35 S]methionine-labeled viral proteins was analyzed by SDS-PAGE. Viral proteins are indicated to the left of the gel image. Lane 1 is the positive control with cytoplasmic extract mock depleted and incubated with poliovirus virion RNA (vRNA). The addition of guanidine hydrochloride (GuHCl) in lane 2 has no effect on translation. Poly(rC) is added in lanes 3 through 9, with increasing concentrations of added PCBP2 wild type (lanes 4 to 6) or uncleavable PCBP2 (QS→ID) (lanes 7 to 9). (B) Quantitation of viral translation was performed using Quantity One software to determine the concentration of the VP3 band (optical density per mm 2 ). A ratio of the determined optical density with the addition of wild-type PCPB2 (white) or PCBP2 (QS→ID) (hatched) to the optical density for poly(rC) was calculated to determine the fold change. The positive controls indicating levels of translation in the absence of poly(rC) RNA or recombinant protein (no protein) are depicted as black columns. Results shown are representative of two independent experiments.
Techniques Used: In Vitro, Recombinant, Labeling, SDS Page, Positive Control, Incubation, Quantitation Assay, Software, Concentration Assay