Total RNA was isolated from the young leaf tissues or roots of control and salt-treated (200 mM NaCl)/ABA-treated (100 μM)/PEG-treated (20%)/cold-treated (4°C) seedlings of IR-29, Pokkali, and Nonabokra [13 (link)]. RNA samples were treated with RNase-free DNase I (Boehringer Mannheim). The cDNA was synthesized using 5 μg of total RNA. The semiquantitative RT-PCR experiments were conducted in triplicates using GoTaq qPCR Master Mix (Promega) with actin as standard and other gene-specific primers in a CFX-96 Bio-Rad thermocycler (Bio-Rad). Increasing temperature from 55°C to 95°C was used for melt curve analysis. The untranscribed RNA was also run as negative control. The relative difference in expression for each sample in individual experiments was determined by normalizing the Ct value for each gene against the Ct value of actin and was calculated relative to a calibrator using the equation 2−ΔΔCt [14 (link)]. The data was imported in TM4 microarray software suite [15 (link)], normalized using GC-RMA algorithm to generate the heat map. Average of three biological replicates was used to get each expression value. The RT-PCR with actin gene was performed as an internal control to normalize the data.
Rnase free dnase 1
Manufactured by Roche
Sourced in United States, Switzerland, Germany, Japan, China, Netherlands
RNase-free DNase I is a laboratory enzyme used for the removal of DNA from RNA samples. It functions by cleaving and degrading double-stranded and single-stranded DNA molecules, while leaving RNA molecules intact.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (95 mentions)
Biotin rna labeling mix, by Roche (72 mentions)
Streptavidin agarose bead, by Thermo Fisher Scientific (55 mentions)
T7 rna polymerase, by Roche (52 mentions)
Trizol reagent, by Thermo Fisher Scientific (46 mentions)
T7 sp6 rna polymerase, by Roche (9 mentions)
Trizol, by Thermo Fisher Scientific (9 mentions)
T7 rna polymerase, by Takara Bio (8 mentions)
Rneasy mini kit, by Roche (6 mentions)
M mlv reverse transcriptase, by Promega (5 mentions)
Orbitrap velos pro lc ms system, by Thermo Fisher Scientific (5 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Iq sybr green supermix, by Bio-Rad (5 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (5 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (5 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (4 mentions)
Pgem t, by Promega (4 mentions)
Bcabest rna pcr kit, by Takara Bio (4 mentions)
Iq5 multicolor real time pcr detection system, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
255 protocols using rnase free dnase 1
1
Semiquantitative RT-PCR Analysis of Stress-Induced Gene Expression
2
Tissue RNA Isolation and Quantification
3
Quantitative Real-Time PCR Analysis
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Total RNAs were extracted from cells using Trizol solution (Thermofisher), according to the manufacturer’s instructions. The quality and quantity of RNAs were assessed by denaturing agarose gel electrophoresis and by spectrophotometry analysis (NanoDrop Technologies), after RNAse-free DNAse-I treatment (Boehringer Mannheim, Indianapolis, IN, USA). RNA was reverse transcribed with SuperScript III (Thermofisher) using 500 ng of total RNA. Quantitative RT-PCR analysis was performed as previously reported [4 (link)]. Glyceraldehydes-3-phophate dehydrogenase (GAPDH) or peptidylprolyl isomerase A (PPIA) were used as housekeeping control genes [18 (link)].
4
RNA Extraction from Frozen Tumor Tissue
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Total tissue RNA was isolated from frozen tumor tissues by use of the TRI Reagent (Molecular Research Center Inc, Cincinnati, OH, USA) according to the manufacturer's protocol. Potentially contaminating DNA was removed by treating with RNase-free DNase I (Boehringer-Mannheim, Mannheim, Germany), followed by phenol/chroloform extraction. RNA was dissolved in RNase-free H2O. The resulting RNA concentration was measured spectrophotometrically (DU500 Spectrophotometer, Beckman Coulter, Fulleron, CA, USA), and the quality of the RNAs was checked by electrophoresis on 1.5% agarose gel.
5
RNA Purification and cDNA Synthesis
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Prior to reverse transcription, RNA samples were subjected to DNase treatment. It was carried at 37 °C for 30 min. in a 10 μl solution containing 1 μg of total cellular RNA, 1× reaction buffer (10 mM Tris–HCl pH 7.5, 2.5 mM MgCl2, 0,1 mM CaCl2), 10 U of ribonuclease inhibitor (MBI Fermentas) and 5 U of RNase-free DNase I (Boehringer Mannheim). To stop the reaction, the samples were supplemented with 1 μl of 25 mM EDTA and incubated in 65 °C for 10 min.
Resulting RNA preparation was directly used in reverse transcription set up with components and procedure of the First Strand cDNA Synthesis Kit (MBI Fermentas). This reaction was primed using either gene-specific oligo R (see below) or random hexamers from the kit. The former was used to produce cDNA for Pfu-driven amplification, the latter—to yield template for real-time PCR.
Resulting RNA preparation was directly used in reverse transcription set up with components and procedure of the First Strand cDNA Synthesis Kit (MBI Fermentas). This reaction was primed using either gene-specific oligo R (see below) or random hexamers from the kit. The former was used to produce cDNA for Pfu-driven amplification, the latter—to yield template for real-time PCR.
6
Affinity-Based Protein-RNA Interactome
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Biotin‐labeled RNAs were transcribed in vitro with the Biotin RNA Labeling Mix (Roche, Mannheim, Germany) and T7 RNA polymerase (Roche), treated with RNase‐free DNase I (Roche), and purified with RNeasy Mini Kit (Qiagen, Hilden, North Rhine‐Westphalia, Germany). Protein‐RNA interactions were carried out using 1 mg whole‐cell lysates and 3 μg of purified biotinylated transcripts for 1 hour at 25°C. Complexes were isolated with streptavidin agarose beads (Invitrogen). The beads were washed briefly thrice and boiled in SDS buffer, and the retrieved protein was detected by mass spectrometry or standard western blotting procedures. Briefly, proteins were extracted from cells using RIPA lysis buffer (Yeason), digested into peptides, and then protein identification was performed using the mass spectrometer (Thermo Fisher Scientific).
7
Identifying lnc-ORA Binding Targets of miR-532-3p
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A miRNA pull-down assay was performed as previously reported (51 (link)). C2C12 cells were transfected with biotinylated miR-532-3p: wildtype miR-532-3p (miR-532-3p-Bio) or mutated miR-532-3p (G–C mutation in the lnc-ORA–binding sites, miR-532-3p-MutBio) and biotinylated control (negative control-Bio) when 70 to 80% of confluency was reached and induced into myogenic differentiation at full confluence. Six days after induction, cell lysates were collected and incubated with Dyna M-280 streptavidin magnetic beads (#11205D; Thermo Fisher Scientific). The product was then treated with RNase-free DNase I (Roche), and RNA was purified using an RNeasy Mini Kit (QIAGEN). The enrichment of lnc-ORA was detected through qRT-PCR.
8
Biotin-labeled RNA Binding Assay
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Biotin-labeled RNAs were transcribed with the Biotin RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN, USA) and T7 RNA polymerase (Roche Diagnostics, Indianapolis, IN, USA), treated with RNase-free DNase I (Roche Diagnostics, Indianapolis, IN, USA), and purified with a RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Subsequently, 1 mg whole-cell lysate from N27 dopaminergic neurons was incubated for 1 h with 3 μg purified biotinylated transcripts at 25°C. Complexes were extracted with streptavidin agarose beads (Invitrogen Inc., Carlsbad, CA, USA). The retrieved protein was detected using the conventional western blot assay.
9
Biotin-labeled RNA Pull-down Assay
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RNA pull-down assay was performed as previously described [22 (link)]. In brief, PCGEM1 or PCGEM1-mut were in vitro transcribed, respectively, and biotin-labeled with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). 1 mg of whole-cell lysates were incubated with 3 μg of purified biotinylated transcripts for 1 h at 25 °C, then the complexes were isolated with streptavidin agarose beads (Invitrogen). The RNA present in the pull-down material was detected by qRT-PCR analysis.
10
Quantitative Analysis of Gene Expression
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The tissues (100 mg each sample) including liver, spleen, kidney, heart, lung, brain, testis, crureus, gluteus and dorsal muscle were collected from above 5 transgenic and 1 control animals. The samples were homogenized in TRIZOL reagent (Invitrogen, USA), and total RNA was isolated according to the manufacturer’s instruction. Total RNA was incubated with RNase-free DNase I (Roche, Switzerland) to eliminate contaminated genomic DNA before being reverse transcribed into cDNA using random hexamer primers and M-MLV Reverse Transcriptase (Promega, USA).The real-time fluorescence quantitative reverse transcription polymerase chain reaction method (qRT-PCR) was established in a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). All the qRT-PCR experiments were in triplicate. β-actin was used as an internal control. The primers were shown in Table 2 . The program and system of qRT-PCR were described previously [19 (link)].
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