Total RNAs were extracted by RNeasy plus mini kit (Qiagen, Hilden, Germany), and the quality and concentration were determined by agarose electrophoresis. Then, RNAs were respectively reverse-transcribed with Goldscript cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer. Semi-quantitative RT-PCR was performed as described previously31 (link) in a volume of 25 μl as follows: 94 °C for 4 min, 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min for 33 cycles followed by 72 °C for 10 min. Real-time quantitative PCR was performed by CFX96 Optics Module (Bio-Rad, Singapore) with SYBR Green I Dye as described previously.32 (link) All results were analyzed according to a previous report.33 (link)β-Actin was used as the internal control gene.
Goldscript cdna synthesis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Goldscript cDNA Synthesis Kit is a laboratory tool designed for the reverse transcription of RNA to complementary DNA (cDNA). The kit includes reagents and instructions necessary for the conversion of RNA into first-strand cDNA, which can then be used in various downstream applications such as PCR, qPCR, or library preparation.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy plus mini kit, by Qiagen (2 mentions)
Easypure rna kit, by Transgene (2 mentions)
Cfx96 optics module, by Bio-Rad (1 mentions)
Peasy blunt zero cloning vector, by Transgene (1 mentions)
Sybr premix ex taq 2 mix, by Takara Bio (1 mentions)
Abi 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Sybr green kit, by Roche (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Sv total rna isolation system, by Promega (1 mentions)
6 protocols using goldscript cdna synthesis kit
1
Quantitative RNA Expression Analysis
2
Cloning and Sequence Analysis of Gshdz4
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The cDNA (full-length) of Gshdz4 was obtained by homologous cloning. The total RNA of G. soja G07256 was isolated from leaves by EasyPure RNA Kit (Transgen Biotech) and was converted to cDNA by reverse transcription using GoldScript cDNA Synthesis Kit (Invitrogen). The full-length cDNA of Gshdz4 was amplified using gene-specific primers: 5′-AAGCCAGTGAAGAGCGAGCGAGA-3′ and 5′-CTACCATTTCGGGTCAAATTGCAAA-3′. The PCR product was cloned into the pEASY-Blunt Zero Cloning Vector (Transgen Biotech) and subjected to sequencing. Sequence alignments were performed using DNAMAN. Phylogenetic analysis was carried out with MEGA 4.0. Similarity of sequences was verified by the BLASTP at NCBI online (http://blast.ncbi.nlm.nih.gov/Blast.cgi ). Homology searches were done by Phytozome (http://www.phytozome.net/soybean ).
3
Analyzing Histone and Housekeeping Gene Expression
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Total RNAs were extracted by RNeasy Plus Mini Kit (Qiagen). One microgram of DNase-treated total RNA was transcribed reversely using Goldscript cDNA Synthesis Kit (Invitrogen), as described by the manufacturer. The cDNA templates of replication dependent histones were transcribed by random primer, and others were transcribed by oligo(dT) primer. RT-PCR and qPCR were performed as described previously (Huang et al. 2009 (link); Zhong et al. 2014 (link)). β-actin or ef1α was selected as housekeeping gene (Livak and Schmittgen 2001 (link)). The primers are listed in Supplemental Table S5 .
4
Quantitative Gene Expression Analysis
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Total RNA was extracted from G. soja or Arabidopsis seedlings by using EasyPure RNA kit (Transgen Biotech), and cDNA synthesis was performed using the GoldScript cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA). cDNA quality was assessed by PCR using specific primers for GAPDH (glyceraldehyde-3-phosphate dehydrogenase, accession no. DQ355800) to exclude genomic DNA contamination. The qRT-PCR was performed in 96-well (25 μL) format using the SYBR Premix ExTaq™ II Mix (TaKaRa, Shiga, Japan) on an ABI 7500 sequence detection system (Applied Biosystems, USA). One microliter of each synthesized cDNA (diluted 1:5) was used as template. Amplification of GAPDH in G. soja or ACTIN2 in Arabidopsis was used as controls. Expression levels for all candidate genes were determined using the 2–ΔΔCT method. The relative transcript levels were calculated and normalized as described previously [53 (link)]. cDNA from three independent plants were treated as three biological replicates and each cDNA sample was repeated three times as three technical replicates, and the results from one representative experiment are shown.
5
EGFP Expression Quantification Protocol
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EGFP uorescence was detected 48 hours post-transfection by uorescence microscopy at 100× magni cation. After cells were digested with pancreatin (PAA Laboratories), each well was suspended in PBS and the average uorescence intensity of each well was measured using ow cytometry. Total RNA was extracted from each well using an RNA total kit (Invitrogen), then two micrograms of RNA were reverse transcribed using a GoldScript cDNA synthesis kit (Invitrogen) with random primers. Quantitative real-time PCR (qRT-PCR) was performed using a SYBR GREEN kit (Roche,NY, USA) according to manufacturer's instructions, with primers EGFP-FP and EGFP-RP to amplify EGFP from PK and DF-1 cells, on a Roche LightCycler 480 system (Roche). Pig GAPDH and Chicken beta-actin were used as controls for PK-15 and DF-1cells, respectively. Standard curves using the two control genes were constructed to assist with actual e ciency calibration, though relative gene expression was calculated for the EGFP gene transcription. Each reaction was performed in triplicate.
6
Zebrafish RNA Extraction and Analysis
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Total RNAs were isolated from zebrafish embryos or adult tissues by using SV Total RNA Isolation System (Promega). The quantity and quality were determined by agarose electrophoresis and spectrophotometer 17 . The isolated RNAs were then reverse-transcribed by using the Goldscript cDNA Synthesis Kit (Invitrogen) following the manufacturer's recommendation. β-actin was detected as an internal control. Semi-quantitative RT-PCR and real-time PCR analyses were performed as described previously 18 (link), and the sequences of primers were given in Supplementary Table S1 .
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