Genomic DNA was extracted from coleoptiles of five-day-old seedlings, using the cetyltrimethylammonium bromide method [31 (link)]. Total RNA was isolated from coleoptiles of five-day-old seedlings with the TIANGEN RNAprep Pure Plant Kit (Tiangen Company, Beijing, China). Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific, Wilmington, DE, USA) and 1% agrose gel. cDNA was obtained from total RNA using the Thermo RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, St. Louis, MO, USA).
Thermo revertaid first strand cdna synthesis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The Thermo RevertAid First Strand cDNA Synthesis Kit is a reagent kit designed for the reverse transcription of RNA into complementary DNA (cDNA). The kit includes all the necessary components to perform this process, including a reverse transcriptase enzyme, primers, and buffers.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Sybr select master mix, by Thermo Fisher Scientific (2 mentions)
Piko real 96 pcr system, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Toyobo (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Plant genomic dna kit, by Tiangen Biotech (1 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Thermo maxima sybr green qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Cfx96 cycler, by Bio-Rad (1 mentions)
Nanophotometer, by Implen (1 mentions)
9 protocols using thermo revertaid first strand cdna synthesis kit
1
Coleoptile Genomic DNA and RNA Isolation
2
Total RNA Extraction from Coleoptiles
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Total RNA was isolated from the coleoptiles of five-day-old seedlings using the TIANGEN RNAprep Pure Plant Kit (Tiangen Company, Beijing, China). The total RNA of each sample was quantified and qualified with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific, Wilmington, DE, USA), and 1% agrose gel. cDNA was obtained from the total RNA using the Thermo RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, St. Louis, MO, USA).
3
Transcription Analysis of Silkworm Larvae Exposed to Baculovirus
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Total RNA was extracted from whole larvae treated with 106 OBs/larva using the Trizol reagent (Invitrogen), as per the manufacturer’s protocol. Whole larvae samples were separately collected from silkworms in the TG-A, TG-B, TG-C, and WT groups treated with 106 OBs/larva (n = 6 per group) harvested for analysis every 12 h from 0 to 72 hpi. RNA was quantified by spectrophotometry and purity was evaluated by gel electrophoresis. First-strand cDNA was prepared with the Thermo RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. To quantify the relative transcription levels of targeted genes, gene-specific primer sets were designed and used for qPCR performed with 2× SYBR Green PCR Master Mix (Toyobo). The housekeeping gene Rp49 (GenBank accession number AB048205.1) [28 (link)] was used as an internal control to standardize the variance of the different templates, and analyzed using the 2−△△CT method [29 (link)]. The mRNA measurements were quantitated in three independent biological replicates and three independent technical replicates. Primers are listed in Supplementary Table S1 .
4
Extraction and Analysis of Aleurone Layer RNA
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Genomic DNA was extracted from fresh leaves 22 days post-anthesis using a plant genomic DNA kit (Tiangen Biotech, Beijing Co. Ltd., Beijing, China). At 14, 22, 28, 35 days post-anthesis (dpa), the aleurone layer of the immature grains was carefully stripped and immediately placed in liquid nitrogen for total RNA extraction, with replicates of 10 grain aleurone layers for each material. Total RNA was extracted using the Tiangen RNAprep Pure Plant Kit (Tiangen Corporation, Beijing, China) according to the standard protocol. RNA concentration and purity were measured using a NanoDrop 2,000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, United States). cDNA was obtained from total RNA using the Thermo RevertAid First Strand cDNA Synthesis Kit (Thermo-Fisher Scientific, Shanghai, China).
5
RNA Extraction and cDNA Synthesis from Aleurone Tissue
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DNA was isolated from 1 g of 10-day-old seedlings according to the methods of Yan et al. [25 ]. At 14 days after anthesis, the aleurone was stripped from one grain carefully, and immediately placed into liquid nitrogen. The aleurones of 20 grains were collected for total RNA extraction. Total RNA was extracted using the Tiangen RNAprep Pure Plant Kit (Tiangen Corporation, Beijing, China) according to the standard protocol. The quality of the total RNA was checked by electrophoresis in a 1.0% agarose gel, and the concentration of total RNA was determined using a NanoDrop (Thermo Scientific, Wilmington, DE, USA). cDNA was obtained from total RNA using the Thermo RevertAid First Strand cDNA Synthesis Kit (Thermo-Fisher Scientific , Shanghai, China).
6
Reference Gene Expression Profiling
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The first strand cDNA was synthesized with 1 μg of high-quality total RNA and 1 μL of oligo dT primers (50 μmol/L) for each sample according to the Thermo RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). The RT-qPCR assays were carried out with a CFX-1000 PCR apparatus and SYBR Premix Ex TaqII (Wuhan Khayal Bio-Technology Company, Wuhan, China) to test the transcription variability of the 10 candidate reference genes across the samples. The total reaction volume was 20 μL, containing 1 μL of diluted cDNA, 10 μL of 2× Ultra SYBR Mixture, 0.4 μL of each primer (10 μmol/L) and 8.2 μL of RNase-free water. The three steps of RT-qPCR program began with 95 °C for 10 min, 45 cycles of 95 °C for 15 s and 60 °C for 15 s, followed by 72 °C for 10 min. The dissociation curve was obtained by heating the amplicon from 56 °C to 95 °C. Each RT-qPCR reaction was carried out with three technical replicates and a template-free control.
7
RNA Extraction and qRT-PCR Analysis
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The total RNA was extracted from frozen villi samples using Trizol reagent, and its purity was evaluated using a Nanophotometer (Implen, Germany). Following reverse transcription with the Thermo RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, United States), 10μl cDNA was amplified using Thermo Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific, United States) on the CFX96 cycler (Bio-rad, United States). The RT-PCR parameters were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 34 s, and 95°C for 15 s. All primers were designed and synthesized by Sangon Bioengineering Company (Table 1 ). The expression levels of the target genes were calculated by the (2–ΔΔCT) method.
8
Quantitative Gene Expression Analysis
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HRMECs were lysed with TRIzol (Invitrogen, Carlsbad, CA, USA), and total RNA was extracted according to the manufacturer's protocol. The concentration and integrity of total RNA were detected with UV spectrophotometry (NANODROP 2000C, Thermo, USA). Reverse transcriptase reactions were performed using a Thermo Revert Aid TM First Strand cDNA Synthesis Kit (K1622, Thermo, USA). Real-time PCR reactions were performed with 2 × SYBR Select Master Mix (Cat. number 4472908, Invitrogen, USA) using a real-time PCR system (Piko Real 96 PCR system, Thermo Scientific, USA). Each sample was measured in triplicate wells. The target gene primers are shown in Table 2 . Data were normalized to the housekeeping genes human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We calculated the changes in mRNA expression according to the 2−ΔΔCT method, with ΔCT = CTTarget gene − CTGAPDH and ΔΔCT = ΔCTTreatment − ΔCTControl. Each experiment was repeated at least three times.
9
Quantitative Analysis of miR-503 and Genes
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In terms of the manufacturer's protocol, TRIzol (Invitrogen, Carlsbad, CA, USA) was added to the HMEC-1 cells for lysis and total RNA was extracted. Total RNA concentration and integrity were determined by UV spectrophotometry (NANODROP 2000C, Thermo, USA). The reverse transcriptase reaction was carried out using a Thermo Revert AidTM First Strand cDNA Synthesis Kit (K1622, Thermo, USA). qPCR reactions were performed using 2 × SYBR Select Master Mix (Invitrogen, USA) and a Real-time PCR system (Piko Real 96 PCR system, Thermo Scientific, USA). Each sample was measured in three wells. The data was normalized to the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6. The relative expression of miR-503 and mRNA of genes were calculated and quantified using 2−ΔΔCt method.
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