RNA was extracted from wheat leaf samples infected with WT Ptr at 1, 3, 5 and 7 days post‐inoculation in triplicate. The second leaf was collected and immediately snap‐frozen. Whole leaf material was ground in liquid nitrogen and subjected to RNA extraction using TRIzol reagent and Direct‐zol RNA miniprep kit (Zymo Research, Irvine, CA, USA). Extracted RNA was treated with TURBO DNA‐free kit (Thermo Fischer Scientific, Waltham, MA, USA) and subjected to reverse transcription using the iScript cDNA synthesis kit (Bioline, London, UK) as per manufacturers' protocols. Quantitative RT‐PCR analysis was performed using Quantitect SYBR Green RT‐PCR kit (Qiagen, Valencia, CA, USA) as described by Rybak et al. (2017). Primers TritA_F and TritA_R were used to amplify the 150 bp region of TtcA (Supporting Information Table S6 for primers). To normalize the gene expression, primer set Act1F4 and Act1R4 were used to amplify the housekeeping gene actin. Samples were analysed with three biological replicates and two‐technical replicates.
Iscript cdna synthesis kit
Manufactured by Meridian Bioscience
Sourced in United Kingdom, United States
The IScript cDNA synthesis kit is a laboratory tool used for the conversion of RNA into complementary DNA (cDNA). It provides the necessary reagents and enzymes to perform this reverse transcription process.
Automatically generated - may contain errors
Lab products found in correlation
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Quantitect sybr green rt pcr kit, by Qiagen (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Chromo4 real time pcr detector, by Bio-Rad (1 mentions)
Illutra rnaspin mini rna isolation kit, by GE Healthcare (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Rneasy mini kit, by Bio-Rad (1 mentions)
Sensifast sybr no rox kit, by Meridian Bioscience (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Cfx manager software, by Bio-Rad (1 mentions)
3 protocols using iscript cdna synthesis kit
1
Quantitative RT-PCR Analysis of TtcA Gene in Wheat
2
Cardiac Differentiation Transcriptome Analysis
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After 7 and 21 days of cell cultivation, total ribonucleic acid (RNA) obtained from the basal and the cardiogenic induced groups (n=5) were prepared using an Illutra RNAspin Mini RNA Isolation kit (GE Healthcare, LittleChalfont, UK). Complementary DNA (cDNA) was synthesized using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions. The RT-qPCR was performed using SsoFast™ EvaGreen® Supermix (Bio-Rad, Singapore) on a Chromo4™ Real-Time PCR Detector (Bio-Rad, USA) with gene specific primers (Table I ). The PCR mix contained SsoFast™ EvaGreen® Supermix (Bio-Rad, Singapore), cDNA template, primers, and nuclease free water to reach a final volume of 20 µl. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control gene for normalization. A melt curve analysis was performed at the end of the reaction and the profiles were obtained by plotting relative gene expression levels of the both basal and cardiogenic induced groups.
3
Quantitative Real-Time PCR Analysis of Kcnh2
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Total RNA extracted from NRVMs and from neonatal rat cardiac fibroblasts (maintained in NRVM medium) using the QIAGEN RNeasy Mini kit was reverse transcribed with the Bio-Rad iScript cDNA synthesis kit, and the resulting cDNA was amplified by polymerase chain reaction using the Bioline SensiFAST SYBR No-ROX kit. Polymerase chain reaction amplifications of rat Kcnh2-and rat 18S rRNA (Rn18s)specific cDNA (for primer sequences, see Data Supplement) were performed in a Bio-Rad CFX96 Touch Real-Time PCR detection system using a 2-step cycling protocol (20-40 cycles of 95°C 10 s, 60°C 30 s) after a 5-minute incubation at 95°C. Quantitative analyses were based on the 2 -ΔΔCT method using Bio-Rad CFX Manager software.
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