Quantitative PCR analysis was performed as previously reported6 (link). Total RNAs were extracted with Trizol (Invitrogen; Carlsbad, CA) and then reverse-transcribed using the HyperScriptTM Reverse Transcriptase (Geneall, Seoul, South Korea) in a PTC-200 Thermal Cycler (MJ Research, Reno, NV). The resulting cDNA (25 ng) was amplified using LaboPassTM SYBR Green Q Master (Cosmogenetech, Seoul, South Korea) for quantitative real-time PCR. PCR experiments were performed in triplicate. Primer sequences are provided as Supplementary Table 1 . Real-time PCR was performed in a CFX ConnectTM Real-Time PCR Detection System (Bio-Rad Laboratories; Hercules, CA). The expression of gene transcripts was normalized to the geomean values of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succineate dehydrogenase complex subunit A, flavoprotein (FP)(SDHA), and hypoxanthine phosphoribosyltransferase 1 (HPRT1)48 (link), and relative expression values were calculated according to the ΔΔCt method.
Hyperscript reverse transcriptase
Manufactured by GeneAll
Sourced in Cameroon
Hyperscript™ Reverse Transcriptase is a laboratory enzyme used for the synthesis of complementary DNA (cDNA) from RNA templates. It catalyzes the reverse transcription process, converting single-stranded RNA into double-stranded cDNA.
Automatically generated - may contain errors
Lab products found in correlation
Amplification reagent kit, by Ampliqon (3 mentions)
Ptc 200 thermal cycler, by Bio-Rad (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Pcr master mix, by GeneAll (1 mentions)
Realq plus 2x master mix for probe, by Ampliqon (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Xp cycler, by Bioer (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Taq dna polymerase, by Ampliqon (1 mentions)
Rotor gene q, by Qiagen (1 mentions)
Hybrid r kit, by GeneAll (1 mentions)
Random primer, by Thermo Fisher Scientific (1 mentions)
9 protocols using hyperscript reverse transcriptase
1
Quantitative PCR Analysis of Gene Expression
2
Cardiac CaMKIIδ Isoforms Quantification
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The total RNA was obtained from 100 mg of the left ventricular frozen tissue using a kit (Gene all, South Korea, Cat no 305-101), in accordance with the manufacturer’s instructions. RNA concentration was verified by spectrophotometric measurement of the absorbance at 260–280 nm and determined by mixture of Tris base, acetic acid, and EDTA(TAE)-agarose gel electrophoresis.
Reverse transcription (RT) was performed using hyperscriptTM Reverse Transcriptase (Gene All, South Korea). RT-PCR was performed using an amplification reagent kit (Ampliqon, Denmark) by the XP-Cycler instrument (TCXPD, Bioer, USA) with CaMKIIδtotal, CaMKIIδ1, CaMKIIδ2, and the rat glyceraldehydes-3-phosphate dehydrogenase (GAPDH) primers. To amplify the cDNA, the 5’ and 3’ primer sequences (forward and reverse) of the CaMKIIδtotal, CaMKIIδ1, and CaMKIIδ2 designed via the Gene Bank (http://blast.ncbi.nlm.gov/Blast.cgi ) revealed that the primers were gene specific. Furthermore, all the primers were verified using a Gene Runner software (Syngene, Cambridge, UK). Subsequently, the primers (forward and reverse) were synthesized to amplify the cDNA encoding GAPDH as a house keeping gene; the sequences of related primers are presented in Table 1 .
Reverse transcription (RT) was performed using hyperscriptTM Reverse Transcriptase (Gene All, South Korea). RT-PCR was performed using an amplification reagent kit (Ampliqon, Denmark) by the XP-Cycler instrument (TCXPD, Bioer, USA) with CaMKIIδtotal, CaMKIIδ1, CaMKIIδ2, and the rat glyceraldehydes-3-phosphate dehydrogenase (GAPDH) primers. To amplify the cDNA, the 5’ and 3’ primer sequences (forward and reverse) of the CaMKIIδtotal, CaMKIIδ1, and CaMKIIδ2 designed via the Gene Bank (
3
Total RNA Extraction and RT-PCR for ASGV Detection
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Total RNA was extracted from each leaf sample using the method described by [21 ] with minor modifications. Reverse transcription (RT) reactions were done using a cDNA solution synthesis kit (HyperScriptTM Reverse Transcriptase, GeneAll, South Korea) and random hexamer primers. PCR reactions done using specific primers, ASGV-U and ASGV-2 [22 (link)] in the PCR master mix (GeneAll, South Korea). PCR products were electrophoresed in a 1.2% (w/v) agarose gel (containing safe stain) in 1X TAE buffer.
4
RNA Extraction and Real-Time PCR Analysis
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5 ml of blood samples were collected in tubes containing EDTA anticoagulants. According to the manufacturer's instructions, total RNA was extracted using a MACHEREY-NAGEL extraction kit (Germany). The extracted RNAs' integrity, concentration, and purity were confirmed using spectrometry, nanodrop, and electrophoresis. 5μg of total extracted RNA was used for cDNA synthesis with hexamer random primer via HyperScriptTM Reverse Transcriptase (GeneAll, South Korea) in a total volume of 20μl. Real-time PCR was performed using Roche Light cycler 96 (version: 1.1.0.1320, Germany), primers, and specific probes for IL-17A, IL-6, and beta-actin as housekeeping genes. The used primer and probe sequences for Real-Time PCR are shown in Table 1.
The total volume of the reaction was about 25μl, of which 4μl was related to the synthesized cDNA solution, 12.5μl of RealQ Plus 2x Master Mix for probe (Ampliqon, Denmark), 500nM of each forwarding and Reverse primers and 250nM TaqMan probe. The instructions were as follows: A. prewarming step (10 min at 94°C); B. denaturation step (15s at 94°C); C. annealing/extension step (60°C for 60s).
The total volume of the reaction was about 25μl, of which 4μl was related to the synthesized cDNA solution, 12.5μl of RealQ Plus 2x Master Mix for probe (Ampliqon, Denmark), 500nM of each forwarding and Reverse primers and 250nM TaqMan probe. The instructions were as follows: A. prewarming step (10 min at 94°C); B. denaturation step (15s at 94°C); C. annealing/extension step (60°C for 60s).
5
Liver RNA Extraction and RT-PCR Analysis
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Total RNA, according to kit instructions, was taken from 100 mg of liver tissue by means of a kit (Gene All, South Korea, Cat no 305-101). RNA concentration was verified and determined by spectrophotometric measurement of the absorbance at 260-280 nm and TAE-agarose gel electrophoresis, respectively. Reverse transcriptase was performed using Hyperscript™ Reverse Transcriptase (Gene All, South Korea). Realtime polymerase chain reaction (RT-PCR) was carried out using an amplification reagent kit (Ampliqon, Denmark) by the XP-Cycler instrument (TCXPD, Bioer, USA) with PTP1B, HNF4α, FAT/CD36, and the rats’ glyceraldehydes-3-phosphate dehydrogenase (GAPDH) primers. The analyses of 5’ and 3’ primer sequences (forward and reverse) of the PTP1B, HNF4α, and FAT/CD36 designed via the Gene Bank revealed that the primers were gene-specific. The forward and reverse primers verified by Gene Runner software were then synthesized to reinforce the cDNA encoding GAPDH as a housekeeping gene. Primers sequences for target genes and housekeeping gene are shown in Table 1 :
6
RNA Extraction and cDNA Synthesis
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Five milliliters of blood samples was collected in anticoagulant EDTA tubes. Total RNA was extracted using the MN kit (MACHEREY-NAGEL, Germany), according to the manufacturer's instructions. Concentration, integration, and purity of RNA samples were determined by spectrometry, Nano-Drop (Thermo Scientific, Waltham, MA), and gel electrophoresis. Five micrograms of total RNA was used for cDNA synthesis with a random hexamer primer through HyperScript Reverse Transcriptase (GeneAll, South Korea) in 20 μL total reaction mixture.
7
Reverse Transcription and PCR Amplification
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Total RNA of samples was isolated by Total RNA isolation kit (DENA Zist Asia). The cDNA(s) were synthesized using Hyper script reverse transcriptase (Gene All) and oligod (T) 18mer, P.SOS.S-REV1, P.SOS.S-REV2, P.SOS.S-REV3, P.SOS.S-REV4, P.NHX.S.REV1, P.NHX.S.REV2 and P.NHX.S.REV3primers (Table 1 ) and amplified with a combination of primers (Table 2 ). The amplifications were obtained in 30 cycles at defined annealing temperature for each pair of primers using TaqDNA polymerase (AMPLIQON). The process finished after a final extension for 5–15 min at 720C (Fig. 1 ).![]()
Regulation of ion homeostasis by ion Na+/H+ pumps antiporters (SOS1), vacuolar Na+/H+ exchanger (NHX) that salt sensors present at the plasma and vacuolar membranes [32 (link)]
8
RNA Extraction and Expression Analysis in Mouse Cervix and Ears
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RNA was obtained from cervix and ears of WT or Tg(K6b-E/6E7) mice using the Hybrid-R kit (GeneAll, Seoul, South Korea). For estrous cycle analysis, 9-week-old CD1 mice were selected at each estrous cycle phase and the whole cervix dissected. For ear regeneration, 3–4 perforations were done in one ear of 3-month-old CD1 female mice and, 7 days later, both injured and non-injured ears were collected. First-strand cDNA was synthetized using HyperScript Reverse Transcriptase (GeneAll, Seoul, South Korea) and random primers (Invitrogen, Carlsbad, CA, USA). The real-time quantitative PCR was performed using KAPA SYBR FAST mix (KAPA Biosystems, Wilmington, MA, USA) in the presence of specific primers (Supplementary Table S2 ) and the Rotor-Gene Q (Qiagen, Wilmington, MA, USA). Gene expression was evaluated using a ΔΔCt method. The housekeeping gene Rplp0 was used to normalize gene expression levels.
9
Quantification of CaMKIIδ and MHC Isoforms
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Total RNA was extracted using an extraction kit (Gene All, South Korea), according to the manufacturer's protocol. The real-time quantification of the target genes was carried out as described previously in our earlier study (17, 18) . Reverse transcriptase (RT) was presented using Hyperscript™ Reverse Transcriptase (Gene All, South Korea). RT-PCR was performed with cDNA synthesis using an amplification reagent kit (Ampliqon, Denmark) by the XP-Cycler instrument (TCXPD, Bioer, and USA). The relative expression of CaMKIIδtotal and isoforms δ2 and δ3 of CaMKIIδ, as well as MHC-β and α isoforms mRNA were normalized to the amount of GAPDH in the same cDNA sample using the standard curve method. Gene Bank (http://blast.ncbi.nlm.gov/Blast.cgi) was used for planning the primer sequences (forward and reverse) of the target genes confirming with the Gene Runner software (Table 1). The amount of PCR yields was normalized to GAPDH as a housekeeping gene. We used the 2 -(ΔΔCt) . Results were presented as the fold-difference to the relevant controls.
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