Total RNA was extracted using Trizol reagent (Invitrogen Life Technologies, Karlsruhe, Germany) following the manufacturer’s instructions. First-strand complementary DNAs (cDNAs) were synthesized using the RevertAidTM first-strand synthesis kit (Fermentas, Burlington, ON, Canada) and then used as templates for RT-PCR. A threshold cycle for each PCR amplification was subjected to 40 cycles of denaturation at 95 °C for 5 minutes, annealing at 95 °C for 15 seconds, elongation at 60 °C for 30 seconds, and finally followed by a melting curve analysis. Primers for the following genes were used: MMP-10 (forward: 5′-GCAGCCCATGAACTTGGCCACT-3′; reverse: 5′-AGGGACCGGCTCCATACAGGG-3′); MMP-13 (forward: 5′-GATGACCTGTCTGAGGAAGACC-3′; reverse: 5′-GCATTTCTCGGAGCCTGTCAAC-3′); Col X (forward: 5′-CTCCTACCACGTGCATGTGAA-3′; reverse: 5′-ACTCCCTGAAGCCTGATCCA-3′); Col II (forward: 5′-CCAGCTGACCTCGCCACTGA-3′; reverse: 5′-GGGTCCAGGCGCACCCTTTT-3′); Runx2 (forward: 5′-GGTTGTAGCCCTCGGAGAGG-3′; reverse: 5′-GCCATGACGGTAACCACAGTC-3′); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward: 5′-TGAGGCCGGTGCTGAGTATGTCGT-3′; reverse: 5′-GGTCCTTTTCACCAGCAAGC-3′). GAPDH was used to normalize expression levels. The results were analyzed using the comparative ΔΔCT method (2(−ΔΔCT)) for the relative quantification of gene expression.
Revertaid first strand synthesis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
The RevertAid First Strand Synthesis Kit is a tool for the reverse transcription of RNA into complementary DNA (cDNA). It provides the necessary components to synthesize the first strand of cDNA from total RNA or poly(A)+ RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (13 mentions)
Gotaq qpcr master mix, by Promega (10 mentions)
Rnasimple kit, by Tiangen Biotech (7 mentions)
Brilliant sybr green qpcr master mix, by Thermo Fisher Scientific (4 mentions)
Nucleospin rna 2 mini kit, by Macherey-Nagel (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Ethidium bromide, by Carl Roth (2 mentions)
100 pb ladder, by GeneDireX (2 mentions)
Dreamtaq, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Rneasy mini rnasimple kit, by Tiangen Biotech (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Mastercycler ep realplex, by Eppendorf (2 mentions)
Oligo dt primer, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time system, by Bio-Rad (2 mentions)
Sybr green pcr master mixes, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
51 protocols using revertaid first strand synthesis kit
1
Quantitative Gene Expression Analysis
2
Quantitative RT-PCR Analysis of Chondrocyte Gene Expression
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Total RNA was extracted from C28/I2 chondrocytes using TRIzol reagent. One microgram RNA was reverse transcribed to cDNA using the RevertAidTM First-Strand Synthesis kit (Fermentas, Waltham, MA, USA). qRT- PCR analysis was carried out using Fast-Start Essential DNA Green Master Mix (Roche, Germany) on an Applied Biosystems 7000 (ABI). The thermal cycling conditions for all reactions were as follows: activation10 min at 95°C, followed by 40 amplification cycles of 95°C for 10 sec, 60°C for 10 sec and 72°C for 10 sec. Gene expression was calculated according to the 2-ΔΔCt comparative method. The sequences of primers are listed in Table 1 .
3
Generation of Lysosomal Gene Expression Constructs
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RNA was isolated from MEFs or NIH3T3 cells using the Nucleospin RNA plus kit (Catalog no. 740984.5, Macherey-Nagel). Subsequently, cDNA was generated using the RevertAid First Strand Synthesis kit (Catalog no. K1621, Thermo Fisher Scientific) according to the manufacturer’s instructions utilizing random hexamer primers. Downstream RT-PCR amplification of lysosomal/endosomal genes was carried out with the respective set of primers (supplemental Table S14 ). PCR products were purified with the High Pure PCR Product Purification Kit (Catalog no. 11732676001, Sigma-Aldrich) and cloned into the pcDNA3.1 Hygro+ mammalian expression vector (Catalog no. V87020, Thermo Fisher Scientific). Successful cloning and sequence identity for the respective gene’s cDNA was confirmed using Sanger sequencing (Eurofins) for each generated plasmid. The Tspan3-myc expression vector was a kind gift from Paul Saftig, and the Rab5-GFP expression vector was a kind gift from Sergio Grinstein.
4
Quantitative Gene Expression Analysis
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RNA was isolated using the NucleoSpin RNA Kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. RNA content was quantified by spectrophotometry and stored at −80 °C until cDNA synthesis. cDNA was synthesized from 0.5 μg RNA with the RevertAid First Strand Synthesis Kit (Thermo Scientific, Roskilde, Denmark). qPCR was performed using the SYBR Green qPCR Master Mix and run on an Aria MX3000p (Agilent Technologies, Lexington, MA, USA). The expression level of the genes of interest was corrected using the reference gene GAPDH or 18S. Primers are listed in Table 1 .
5
qPCR Transcriptomic Profiling Protocol
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Equal amounts of experimental cells were subjected to isolation of total RNAs by using the RNA simple Kit (Tiangen Biotech Co., Beijing, China). Then, 500 ng of total RNAs was added in a reverse-transcriptase reaction to generate the first strand of cDNA (with the Revert Aid First Strand Synthesis Kit from Thermo, Waltham, MA, USA). The synthesized cDNA fragments were served as the template for qPCR, in the GoTaq®qPCR Master Mix (from Promega), before being deactivated at 95°C for 10 min, and then amplified by 40 reaction cycles of the annealing at 95°C for 15 s and then extending at 60°C for 30 s. The final melting curve was validated to examine the amplification quality, whereas the mRNA expression level of β-actin served here as an optimal internal standard control. All the primers used for qPCR (Table S1 ) were synthesized by Tsingke (Chengdu, China).
6
Quantitative RT-PCR Protocol for Gene Expression
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Total RNA was isolated from TRIzol (Thermo Fisher, Mississauga, ON, Canada), following the manufacturers protocol for small number of cells, using glycogen as a carrier. To remove any residual DNA, pellets were dissolved in water and digested with amplification grade DNAaseI (Sigma Aldridge, St. Louis, MO, USA). cDNA was synthesized using RevertAid First strand Synthesis Kit (Thermo Fisher, Mississauga, ON, Canada), using oligo dT primers. Expression levels of transcripts were assessed by qPCR assay performed in the Mastercycler® (Eppendorf, Mississauga, ON, Canada), using SYBR Green PCR mix (Applied Biosystems, Foster City, CA, USA or Wisent, Saint-Jean-Baptiste, QC, Canada). Amplification conditions for each primer set was optimized for efficiency. Dissociation curves at the end of the reaction were checked for each sample. Fold changes using ΔΔCΤ were generated using β-actin as a housekeeping gene. Primer sequences are listed in Supplementary Table S1 .
7
Comprehensive RNA Isolation and Analysis Protocol
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Total RNA was isolated using Aurum Total RNA Mini Kit (Bio-Rad, cat #732-6820). RNA quality was verified using RNA LabChip Kit (Agilent, RNA 6000 Nano) and concentration measured with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). One microgram of total RNA was converted to cDNA using RevertAid First Strand Synthesis Kit (Thermo Scientific, K1621) and RT2 First Strand Kit (SABiosciences, cat #330401). The level of specific transcripts was assessed using RT2 Profiler PCR Array Human Fibrosis (SABiosciences, cat #PAHS-120) according to the manufacturer's protocols. Confirmation of RT2 Profiler PCR Array was performed by Q-PCR analysis using TaqMan Gene Expression Assays (Applied Biosystems, cat #4331182). All PCR reactions were performed using 7500 Real-Time PCR System (Applied Biosystems). RT2 Profiler PCR Array data were analyzed using RT2 Profiler Array Data Analysis Version 3.5 Software. RT-
RCR data were analyzed using comparative ΔΔCt method. If not specified, GAPDH used as endogenous reference.
RCR data were analyzed using comparative ΔΔCt method. If not specified, GAPDH used as endogenous reference.
8
RNA-seq Validation via qPCR in Wheat Straw Fermentation
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For validation of RNA sequencing results, 1 µg of total RNA isolated as previously described from semi-solid wheat straw fermentation was treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA) and used for cDNA synthesis with the RevertAid First-Strand Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) using oligo (dT) nucleotides. Two-step quantitative PCR (qPCR) reactions were carried out in a rotor-gene apparatus (Qiagen, Hilden, Germany). Each reaction contained 5 µL of QuantiNova SYBR Green PCR MasterMix and 1 µL of 1:8 dilution of cDNA as the template (total volume: 10 µL). The primers used for qPCR, their final concentration on the assay, and the annealing/extension temperature for each primer pair are listed in Supplementary Table S3 . Internal standard genes for relative quantification (sarA and cox5) were selected according to a previous standardization for Aspergillus niger [37 (link)]. Relative expression of sih1, sih2, sih4, and AsHog1 genes was calculated as described by Pfaffl [38 (link)] using the REST software (2009, Qiagen, Hilden, Germany).
9
Multiplex RT-PCR for Stem Cell Markers
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Total RNA was extracted from cells at end of P3 using (RNeasy Mini Kit–Qiagen, Germany). Reverse transcription was carried out using (RevertAid First Strand Synthesis Kit-Thermo fischer, USA) and RT-PCR reaction was performed using (Dream Taq, Thermo fischer, USA) to detect expression of CD44, CD90, CD105, using beta-actin gene expression as a control under the condition detailed in Table 1 .
Primers that were used in RT-PCR reaction are shown in theTable 2 .
2% agarose gel electrophoresis was performed to detect PCR products using 100 pb ladder (GeneDireX, Taiwan) and ethidium bromide (Carl Roth, Germany) to visualize amplicons.
Primers that were used in RT-PCR reaction are shown in the
2% agarose gel electrophoresis was performed to detect PCR products using 100 pb ladder (GeneDireX, Taiwan) and ethidium bromide (Carl Roth, Germany) to visualize amplicons.
10
Quantitative Real-Time PCR Protocol
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Experimental cells were subjected to isolation of total RNAs by using the RNAsimple Kit (Tiangen Biotech Co., Beijing, China). Then, 500 ng of total RNAs were added in a reverse-transcriptase reaction to generate the first strand of cDNA (with the Revert Aid First Strand Synthesis Kit from Thermo, Waltham, MA, USA). The synthesized cDNA was served as the template for qPCR, in the GoTaq® qPCR Master Mix (from Promega,), before being deactivated at 95 °C for 10 min, and then amplified by 40 reaction cycles of the annealing at 95 °C for 15 s and then extending at 60 °C for 30 s. The final melting curve was validated to examine the amplification quality, whereas the mRNA expression level of β-actin served as an optimal internal standard control.
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