RNA-seq was carried out as described previously [34 (link)]. Total RNA from seminiferous tubules from one animal was extracted using 1 ml TRIzol reagent (Life Technologies). Quantity of the isolated total RNA was determined by spectrophotometric measurement using the NanoDrop device. The quality of the RNA was determined using standard 1% agarose gel electrophoresis. For the RT-PCR and quantitative RT-PCR (qRT-PCR), the RNA was treated with RQ1 (RNA Qualified) RNase-Free DNase (Promega) as recommended. RNA-seq libraries from three wild-type and three knockout animals were prepared using Illumina's TruSeq RNA Sample Preparation Kits v2 (Illumina Inc.). Briefly, 4 μg of total RNA was poly(A)-selected, followed by generation of double-stranded cDNA. Adapters were ligated, and resulting fragments were amplified by limited number of PCR cycles. RNA-seq libraries were sequenced on HiSeq 2000 platform with 90-nucleotide coverage of each end (PE90). Over 41 million individual sequences from each library were collected with a Q20% larger than 97%.
Rq1 rna qualified rnase free dnase
Manufactured by Promega
Sourced in United States
The RQ1 (RNA Qualified) RNase-Free DNase is a lab equipment product designed to remove DNA from RNA samples. It is an enzyme that specifically digests DNA while leaving RNA intact, allowing for the purification of high-quality RNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Improm 2 reverse transcription system, by Promega (2 mentions)
Truseq rna sample preparation kits v2, by Illumina (1 mentions)
Syber select master mix, by Thermo Fisher Scientific (1 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Lightcycle 96 system, by Roche (1 mentions)
Sybr green master mix, by Bio-Rad (1 mentions)
Rotor gene q real time pcr cycler, by Qiagen (1 mentions)
M mlv reverse transcription system, by Promega (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Wizard genomic purification kit, by Promega (1 mentions)
5 protocols using rq1 rna qualified rnase free dnase
1
RNA-seq analysis of seminiferous tubules
2
Quantitative PCR Analysis of T. brucei Gene Expression
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T. brucei total RNA was extracted using TRIzol Reagent (Invitrogen). To remove residual DNA, the DNase treatment was performed with RQ1 (RNA Qualified) RNase-Free DNase (Promega) according to the manufacturer's instruction. RNA quantification was performed with NanoDrop One Spectrophotometer (Thermo Fisher Scientific), and 1 μg of RNA was run on a formaldehyde gel to check the integrity of RNA. To prepare templates for quantitative PCR, cDNA was synthesized from 1 μg of RNA by ImProm-II Reverse Transcription System (Promega), using random primers (Invitrogen. Real-time PCR was performed in triplicate with the SYBERSelect Master Mix (Applied Biosystems) on LightCycle 96 system (Roche).
qPCR analysis for TbAPI5, TbFOP, TbNTF2L, and TbHYP was normalized using the data obtained by amplification of 7SL RNA.supplemental Table S3 lists the primers used for qPCR, PCR conditions were as follows: preincubation at 95 °C for 10 min, followed by 65 cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 20 s, and extension at 72 °C for 20 s, one cycle of melting at 95 °C for 60 s, 40 °C for 60 s, 65 °C for 1 s, and 97 °C for 1 s. Expression levels were calculated by Pfaffl’s method (88 ).
qPCR analysis for TbAPI5, TbFOP, TbNTF2L, and TbHYP was normalized using the data obtained by amplification of 7SL RNA.
3
Quantifying Bcsnf1 Gene Expression in Botrytis
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qRT-PCR was performed in order to compare the Bcsnf1 gene expression level of the wild type and the complemented strains in vitro. In vitro, four-day-old mycelium was grown and harvested on MM containing 1% PGA or xylan at pH 5 or pH 7. Samples were frozen immediately in liquid nitrogen and stored at −80 °C until utilization. Total RNA was extracted following the protocol of Reid et al. (2006) [51 (link)]. First-strand cDNAs were synthesized with an ImProm-II™ Reverse Transcription System (Promega, Madison, WI, USA), following the manual’s instructions, then the samples were treated with RQ1 (RNA Qualified) RNase-Free DNase (Promega, Madison, WI, USA). qRT-PCR was performed with SYBR® Green master mix (BIO-RAD, Hercules, CA, USA) on a Rotor-Gene Q real-time PCR cycler (Qiagen, Hilden, Germany) with the Bc-Tub-For, Bc-Tub-Rev; and Snf1-ORF-For, Snf1-ORF-Rev primer pairs (Table S1 ). qPCR conditions were as follows: 40 cycles of 95 °C for 20 s, and 57 °C for 20 s and 72 °C for 30 s. Relative expression of the Bcsnf1 gene compared to the tubulin reference gene was determined using the 2−ΔΔCt method [52 (link)].
4
Quantitative RT-qPCR for ZIKV RNA Detection
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Total cell RNA was extracted by TRIzol™ reagent following the manufacturer’s protocol. Both double- and single-stranded DNA in the RNA samples were denatured by using RQ1 (RNA Qualified) RNase-Free DNase (Promega). The RNA samples were reverse transcribed into complementary DNA by using the M-MLV Reverse Transcription System (Promega). Absolute quantitation of ZIKV RNA copies was conducted relative to the standard curves. RT-qPCR analysis was conducted using serially diluted expression plasmids containing the coding sequence of ZIKV NS5 with the following specific primers: forward primer, 5′-aagcaaaaggtagccgcgcc-3′, and reverse primer, 5′-tgtcccagccagcagtgtca-3′, targeting the ZIKV NS5 gene. The reactions were performed using ABI Step One real-time PCR-system (ABI Warrington, UK).
5
Isolating Biomolecules from Parasites
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Isolated parasites were used as RNA, genomic DNA (gDNA) and total protein source. Total RNA was extracted from the sample using the Trizol method and treated with RQ1 (RNA-qualified) RNase-free DNase (Promega, Madison, USA) according to the manufacturer’s recommendations. SuperScript III enzyme (RT+) (Invitrogen, Carlsbad, USA) was used for synthesising complementary DNA (cDNA) in the following conditions: 65 °C for 5 min, 50 °C for 1 h and 70 °C for 15 min. An additional reaction without the SuperScript III enzyme (RT-) was used as negative control, following 15 min incubation at 37 °C with RNase (Promega). A Wizard Genomic purification kit (Promega) was used for obtaining the gDNA. Regarding protein extraction, the parasites were homogenised in lysis buffer containing 5% SDS, 10 mM PMSF, 10 mM iodoacetamide, 1 mM EDTA and then spun at 16,000× g for 5 min. The proteins were recovered from the supernatant and quantified using a BCA protein assay kit (Thermo Scientific, Rockford, USA). RNA, cDNA, gDNA and total protein were stored at -70 °C until later use.
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