Total RNA was extracted from rose petals using the hot borate method as previously described [12 (link)]. The cDNA templates were synthesized from 1 μg total RNA using the HiScript® II reverse transcriptase kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. qRT-PCR was performed in a Step One Plus real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) using an M5 HiPer real-time PCR super mix (Mei5 Biotech, Beijing, China). The ubiquitin RhUBI2 gene was used as an internal control [34 (link)].
Hiscript 2 reverse transcriptase kit
Manufactured by Vazyme
Sourced in China
HiScript II Reverse Transcriptase Kit is a laboratory tool used for the synthesis of complementary DNA (cDNA) from RNA templates. The kit includes the necessary reagents and enzymes required for the reverse transcription process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Aceq universal sybr qpcr master mix, by Vazyme (4 mentions)
Trizol reagent, by Takara Bio (3 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (3 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (3 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (2 mentions)
Sybr green master mix high rox premixed, by Vazyme (2 mentions)
Aceq qpcr sybr green master mix, by Vazyme (2 mentions)
Trizol regent, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Selleck Chemicals (2 mentions)
Mirna 1st strand cdna synthesis kit, by Vazyme (2 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (2 mentions)
E z n a plant rna kit, by Omega Bio-Tek (2 mentions)
M5 hiper realtime pcr super mix, by Mei5 Biotechnology (2 mentions)
Sybr green, by Roche (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Primestar kit, by Takara Bio (1 mentions)
Sybr green master mixes, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 real time pcr system, by Roche (1 mentions)
38 protocols using hiscript 2 reverse transcriptase kit
1
Rose Petal RNA Extraction and qRT-PCR
2
Quantifying Gene Expression in Mouse Uterine SMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs from mouse uterine SMCs were isolated using TRIzol reagent (TaKaRa), RNA was reversed transcribed into cDNA with HiScript II Reverse Transcriptase kit (Vazyme), and then RT-qPCR was performed using ChamQTM Universal SYBR® qPCR Master Mix (Vazyme) on the CFX96 Touch Real-Time PCR Detection system (BioRad). All reactions were run in triplicate. Table 1 shows the primer sequences. The specificity of PCR product amplification was assessed by analysis of the melting curve. Gene expression was normalized with housekeeping genes, ribosomal protein L7 (Rpl7). As both genes were stable, the ΔΔCT method was employed to determine relative changes in gene expression compared to Rpl7.
3
Capturing and Characterizing Phosphoglucomutase in Gracilaria lemaneiformis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The whole genome sequences of G. lemaneiformis were captured from our own laboratory database [38 (link)]. For catching the PGM protein sequence, the Hidden Markov Model (HMM) analysis was used for the search. The HMM profiles of the phosphoglucomutase/phosphomannomutase (PF02878, PF02879, PF02880 and PF00408) from the Pfam protein family database Pfam version 35.0 (http://pfam.xfam.org/ , 29 June 2022) were used as the query to search the G. lemaneiformis protein database with an e-value ≤ e−10. Blastp was subsequently used to search for missing possible PGM candidates. Pfam and SMART (http://smart.embl-heidelberg.de/ , 29 June 2022) were then used to confirm the conserved domain, and the protein sequences lacking the conserved domains were excluded.
The total RNA from G. lemaneiformis was isolated by a RNeasy Plant mini kit (QIAGEN, Germany), according to the manufacturer’s protocol. The cDNA for the full-length sequence cloning was subsequently synthetized by using HiScript II Reverse Transcriptase Kit (Vazyme, Nanjing, China). The opening reading frames (ORFs) of three possible GlPGM were annotated as GlPGM1, GlPGM2 and GlPGM3, and finally amplified by PCR, using Prime Star Kit (Takara, Dalian, China). The gene-specific primers with different restriction endonuclease site are indicated in the additional fileTable S1 (Supplementary Materials) .
The total RNA from G. lemaneiformis was isolated by a RNeasy Plant mini kit (QIAGEN, Germany), according to the manufacturer’s protocol. The cDNA for the full-length sequence cloning was subsequently synthetized by using HiScript II Reverse Transcriptase Kit (Vazyme, Nanjing, China). The opening reading frames (ORFs) of three possible GlPGM were annotated as GlPGM1, GlPGM2 and GlPGM3, and finally amplified by PCR, using Prime Star Kit (Takara, Dalian, China). The gene-specific primers with different restriction endonuclease site are indicated in the additional file
4
Quantitative Gene Expression Analysis in Mouse Lung
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from mouse embryonic lungs using TRIzol® Reagent (TaKaRa Bio, Tokyo, Japan) according to the manufacturer’s manual. RNA was converted to cDNA using the HiScript II Reverse Transcriptase Kit (Vazyme Biotech, Nanjing, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green Master Mix-High ROX Premixed (Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions in a Stepone Plus system (BioRad, Los Angeles, CA, USA). Gene expression was calculated using the 2−ΔΔCt method, in which GAPDH was used as an internal reference for each sample. All reactions were performed in triplicate. The primers used for qPCR were shown in Supplementary Table S1 .
5
RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs from tissues and cells were extracted using Trizol reagent (Invitrogen) following the manufacturer’s instructions. cDNAs were prepared using HiScript® II Reverse Transcriptase Kit (Vazyme). SYBR™ Green master mixes (Life Technologies) and Light Cycler® 480 Real-Time PCR System (Roche) were used for quantitative real-time PCR (qRT-PCR) detection. 18s and Gapdh were used for normalization. Primers for qRT-PCR are listed in Supplementary Table 1 .
6
Quantitative Expression Analysis of S. Typhimurium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
S. Typhimurium strains were grown in LB medium, at 37 °C and on a shaker (220 r/min), until OD600 reached 0.8–1.0, and the total RNA was extracted using a BIOG bacterial RNA extraction kit (BAIDAI, Changzhou, China). Cells were cultured in 12-well culture plates at 37 °C with 5% CO2 until they had grown to approximately 80% confluence, and treated with r0306 or eluent solvent for 10 h. The total RNA was extracted using a FastPure® Cell/Tissue Total RNA isolation Kit (Vazyme, Nanjing, China). Reverse transcription of total RNA was conducted using a HiScript® II Reverse Transcriptase Kit (Vazyme, Nanjing, China). Quantitative PCR was operated on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using AceQ qPCR SYBR green master mix (Vazyme, Nanjing, China). The mRNA expression of target genes in host cells was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), while the mRNA expression of target genes in S. Typhimurium was normalized to that of DNA gyrase subunit A (gyrA). The results were calculated using the 2−ΔΔCt method. The sequence information for all primers used in the qRT-PCR is listed in Table S2 .
7
Quantitative RT-PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
First-strand cDNA was synthesized from the total RNA of each sample using the HiScript II Reverse Transcriptase Kit (Vazyme, Nanjing, China). The cDNA was diluted to 100 ng/µL and mixed with TransStart TOP Green qPCR SuperMix (TransGen, Beijing, China) to a total of 20 µL for qRT-PCR. The amplifications were conducted on an ABI Prism 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA, USA) according to the instructions provided by the manufacturer. Each qRT-PCR reaction included three biological replicates and three technical replicates. The expressions levels were normalized using ACTIN (GenBank: AY305733) as an internal reference and calculated using the 2−∆∆Ct method [48 (link)]. The specific primers (Table S6 ) were designed using Oligo 7 software [49 (link)] and synthesized by Sangon Biotech (Shanghai, China).
8
Quantitative Real-Time PCR Analysis of OA FLS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative real‐time PCR analysis (QPCR) was performed as described previously.2 Total cellular RNA was extracted from human OA FLS using TRIzol reagent (Invitrogen), and RNA was quantitated by spectrophotometry at 260 nm using an ultraviolet spectrophotometer (Thermo Scientific NanoDrop 2000). Then cDNA synthesis was performed using HiScript® II Reverse Transcriptase kit (Vazyme Biotech Co). Quantitative real‐time PCR (qPCR) was performed using AceQ® Universal SYBR® qPCR Master Mix (Vazyme Biotech Co). The protocol of real‐time PCR was as follows: 10 min 95°C, followed by 40 cycles of 15 s 95°C and 1 min 60°C. The primer's sequences of the targeted genes are listed in Table 1 . And data were analysed using 2−ΔΔCT method.
9
Wheat TaMGT Gene Expression Under Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The relative expression levels of TaMGT genes in roots and leaves of wheat under Mg2+ deficiency (-Mg2+), aluminum stress (+Al), and abscisic acid (+ABA) treatments were analyzed by qRT-PCR. Total RNA was extracted from roots and leaves using TRIzol reagent (Life, USA). cDNA was reverse transcribed using the HiScript II Reverse Transcriptase kit (Vazyme, Nanjing, China).Three biological replicates were performed for each sample, with three technical replicates repeated each. The qPCR primers used in present study were listed in Supplementary Table 1
10
Isolating and Quantifying Extracellular Vesicle miRNAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TransZol Up Plus RNA Kit (Trans Gen Biotech) was used for extracting total RNA. RNA reverse transcription was performed by using HiScript II Reverse Transcriptase kit (Vazyme). Real-time quantitative PCR (RT-qPCR) was performed in triplicates using an Applied Biosystems 6 Flex (Thermo Fisher).
The miRNAs were extracted from LEVs using TRIzol reagent (Invitrogen). Briefly, the LEVs were redissolved in 250 μl PBS and mixed with 750 μl TRIzol. Given that U6 mRNA may not be encased within LEVs, caenorhabditis elegans miR-39 (cel-miR-39, 5 nM, RuiboBio) was spiked-in for control. To increase small RNA retrieval, Dr.GenTLE™ Precipitation Carrier (TAKARA) was used per manufacturer's instructions. RT-qPCR was used to determine miRNA levels using miDETECT A TrackTM miRNA RT-qPCR Kit (RuiboBio). The primers for all MicroRNA were obtained from RiboBio Company. The PCR primers are shown in Additional file7 : Table S1.
The miRNAs were extracted from LEVs using TRIzol reagent (Invitrogen). Briefly, the LEVs were redissolved in 250 μl PBS and mixed with 750 μl TRIzol. Given that U6 mRNA may not be encased within LEVs, caenorhabditis elegans miR-39 (cel-miR-39, 5 nM, RuiboBio) was spiked-in for control. To increase small RNA retrieval, Dr.GenTLE™ Precipitation Carrier (TAKARA) was used per manufacturer's instructions. RT-qPCR was used to determine miRNA levels using miDETECT A TrackTM miRNA RT-qPCR Kit (RuiboBio). The primers for all MicroRNA were obtained from RiboBio Company. The PCR primers are shown in Additional file
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!