The reverse transcription was performed with TransScript II First-Strand cDNA Synthesis SuperMix (Transgen, AH301-02) using 2 μg total RNA. The qPCR was performed with RealSYBR Mixture (CWBIO, cw0760) on Bio-rad CFX-96 Real-time PCR Instrument. UBI3 (Solyc01g056840) was used as the internal control of RT-qPCR. The relative expression of each gene in the ovary wall sample at 0 DPAe (ow-0 DPAe) was normalized as 1. Primers for RT-qPCR are listed in Supplementary Table S12 .
Transscript 2 first strand cdna synthesis supermix
Manufactured by Transgene
Sourced in China, United States
TransScript II First-Strand cDNA Synthesis SuperMix is a laboratory product designed for the reverse transcription of RNA into complementary DNA (cDNA). It provides a one-step solution for the efficient and reliable synthesis of first-strand cDNA from various RNA templates, including mRNA, total RNA, and viral RNA.
Automatically generated - may contain errors
Lab products found in correlation
Trizol solution, by Transgene (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Cfx96 real time pcr instrument, by Bio-Rad (3 mentions)
Rna easy kit, by Transgene (3 mentions)
Realsybr mixture, by CWBIO (2 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (2 mentions)
Trnzol reagent, by Tiangen Biotech (2 mentions)
Iq5 lightcycler, by Bio-Rad (2 mentions)
Sybr fast qpcr kit, by Roche (2 mentions)
Trizol reagent, by Tiangen Biotech (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Transstart top green qpcr supermix, by Transgene (1 mentions)
Xhoi, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
E z n a gel extraction kit, by Omega Bio-Tek (1 mentions)
Easytaq dna polymerase, by Transgene (1 mentions)
Restriction endonuclease, by New England Biolabs (1 mentions)
Firstchoice rlm race kit, by Thermo Fisher Scientific (1 mentions)
Dnase 1 digestion, by Qiagen (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
25 protocols using transscript 2 first strand cdna synthesis supermix
1
RT-qPCR Analysis of Ovary Wall Genes
2
Quantitative Real-Time PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was extracted using the TRIzol (Life Technologies) method. The isolated RNA was then subjected to reverse transcription using the TransScript II First-Strand cDNA Synthesis SuperMix (Transgene, Beijing, China) [22 (link)]. Quantitative real-time PCR was performed on each cDNA sample with the TransStart Top Green qPCR SuperMix (Transgene, Beijing, China) by Bio-Rad Chromo4 Detection System (Bio-Rad, USA) according to the manufacturer’s protocol [22 (link)]. The measured Ct values were converted to relative copy-numbers using the ΔΔCt method [27 (link)]. Amplification of TUA5 (Glyma05g29000.1) was used as an internal control to normalize all data [22 (link)]. The RNA of 48h LL condition was included in each batch of quantitative real-time PCR for normalization.
3
Isolation and Cloning of P. tricornutum IDH1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RNA isolation, the P. tricornutum cells were harvested by centrifugation at 1500× g and 4 °C for 10 min. Total RNA of P. tricornutum was extracted using the RNAprep Pure Plant Kit (Cat.#DP432, TIANGEN Biotech, Beijing, China) according to the user’s manual. The first-strand cDNA was synthesized from 2 ng of total RNA with TransScript II First-Strand cDNA Synthesis SuperMix (Cat.#AH301-02, TransGen Biotech, Beijing, China) according to the manuals.
According to the coding sequence of IDH1 from the genome of P. tricornutum CCAP1055/1, two pairs of primers (Table S3 ) were designed to amplify the full-length PtIDH1 and shortened (without the sequence encoding MTP, residues 1‒53) genes. Amplification using a polymerase chain reaction (PCR) program was performed as follows: 95 °C for 3 min, 30 cycles of 95 °C for 30 s, 55 °C for 15 s, and 72 °C for 25 s. The PCR products with NdeI and XhoI (Thermo Scientific, Shanghai, China) digestion were cloned into expression vector pET-22b(+). The correct recombinant plasmids, verified by sequencing (General Biosystems, Hefei, China), were introduced into the prokaryotic expression strain E. coli Rosetta (DE3).
According to the coding sequence of IDH1 from the genome of P. tricornutum CCAP1055/1, two pairs of primers (
4
Fungal Genomic DNA Extraction and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fungal genomic DNA was extracted as described previously (10 (link)). Plasmids and PCR products were purified using the E.Z.N.A. gel extraction kit (Omega Bio-Tek, Norcross, GA). Phusion high-fidelity DNA polymerase (New England Biolabs, Ipswich, Massachusetts) and EasyTaq DNA polymerase (TransGen Biotech, Beijing, China) were used for PCRs. Sequences were determined by SinoGenoMax Co., Ltd. (Beijing, China). Restriction endonucleases and DNA-modifying enzymes were from New England Biolabs (Ipswich, Massachusetts).
RNA was extracted with the Qiagen RNeasy minikit, and carryover DNA was removed by DNase I digestion (Qiagen, Valencia, CA). cDNA fragments were synthesized using the TransScript II first-strand cDNA synthesis supermix (TransGen Biotech, Beijing, China). Introns were identified by comparing genomic and cDNA sequences. The 5′ ends of the mRNA and the poly(A) attachment sites were mapped by 5′- and 3′-RACE–PCR (FirstChoice RLM-RACE kit; Ambion, Austin, Texas).
All primers used in the study are listed inTable S3 in the supplemental material.
RNA was extracted with the Qiagen RNeasy minikit, and carryover DNA was removed by DNase I digestion (Qiagen, Valencia, CA). cDNA fragments were synthesized using the TransScript II first-strand cDNA synthesis supermix (TransGen Biotech, Beijing, China). Introns were identified by comparing genomic and cDNA sequences. The 5′ ends of the mRNA and the poly(A) attachment sites were mapped by 5′- and 3′-RACE–PCR (FirstChoice RLM-RACE kit; Ambion, Austin, Texas).
All primers used in the study are listed in
5
Total RNA Extraction and Analysis
6
RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from the LPS-unstimulated and -stimulated cells was obtained by using ice-cold TriZol reagent (TransGene, Shanghai, China) following the manufacturer’s instructions. The concentration of the total RNA is determined by spectrophotometry (BioTek, Winooski, VT) at an optical density of 260 nm. The quality of the RNA is monitored by A260/A280 ratio (1.8∼2.0). Then 2 µg mRNA is reverse-transcribed into cDNA with TransScript II First-Strand cDNA Synthesis SuperMix (TransGene, Shanghai, China). RT-qPCR was carried out on ABI Step One Software System (ABI, Foster City, CA) using HotStart SYBR Green qPCR Master Mix (USB, Cleveland, OH). GAPDH was used as a reference gene. All PCR primers were synthesized by Sangon Biotech (Sangon, Shanghai, China).
7
Circadian Rhythms in Fig Wasp Pollinators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We made field collections of the fig wasp C. solmsi, the pollinator of Ficus hispida, from Danzhou (19°30′ N, 109°29′ E), Hainan province, China between July and August 2012. Adult females and males were collected from naturally growing syconia when the loose female wasps were yet to emerge. (Male wasps do not emerge from the syconium.) We also collected adult females from different syconia. These females were exposed to natural light for at least three additional hours prior to processing.
After collection, all wasps were flash frozen in liquid nitrogen every 3 hr over a 24 h period. We then isolated total RNA from each wasp, with the first cDNA strand synthesized using TransScript II First Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Ultimately, we used a Real Time-qPCR (RT-qPCR) technique to obtain daily transcript levels of timeout. RPL13a and UBC were selected as the reference genes for normalizing the RT-qPCR data15 (link). The detailed methods of samples collection, RNA isolation and cDNA synthesis, and RT-qPCR expression analysis are provided assupplement material .
After collection, all wasps were flash frozen in liquid nitrogen every 3 hr over a 24 h period. We then isolated total RNA from each wasp, with the first cDNA strand synthesized using TransScript II First Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Ultimately, we used a Real Time-qPCR (RT-qPCR) technique to obtain daily transcript levels of timeout. RPL13a and UBC were selected as the reference genes for normalizing the RT-qPCR data15 (link). The detailed methods of samples collection, RNA isolation and cDNA synthesis, and RT-qPCR expression analysis are provided as
8
Mammary Tissue RNA Extraction and Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA from the mammary tissues or cells was extracted using TriZol solution (TransGene, Shanghai, China) according to the manufacturer's instructions. The assessment of the quantity and quality of the total RNA was conducted by a spectrophotometer (NanoDrop Technologies, Wilmington, DE). The first‐strand cDNA was generated from 3 µg total RNA using TransScript II First‐Strand cDNA Synthesis SuperMix (TransGene). Real‐time quantitative PCR (RT‐qPCR) was carried out to examine the levels of genes by iQ5 light cycler (Bio‐Rad, Hercules, CA) in 20 µL reactions. Finally, the expression of each gene was normalized to GAPDH. The primers used are listed in Table S1 .
9
RNA Extraction and cDNA Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from each RNA sample was extracted with TRIzol Reagent (Invitrogen) and treated with RNase-free DNaseI (Invitrogen). A NanoDrop-2000 spectrophotometer (Thermo, Madison, WI, USA) was used to measure the RNA purity (A260/A280) and concentration. The key issue related to this method is the “false positives” generated by genomic DNA contamination, so before reverse transcription, all RNA samples were confirmed to contain no genomic DNA contamination by PCR with the universal Wolbachia wsp 81 F/691R primers [52 (link)] using TransTaq polymerase High Fidelity (TransGen Biotech, Beijing, China), [20 (link), 34 (link)] (Additional file 5 ). First-stranded cDNA was then synthesized from 1 μg of total RNA with random primers [53 (link)] in a 20 μl reaction volume using TransScript II First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). wsp expression was characterized as the positive control to demonstrate the quality of all the cDNA samples (Additional file 5 ).
10
Quantifying Gene Expression in C. pilosula Roots
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA of the C. pilosula roots at different developmental stages was extracted with a Quick RNA Isolation Kit (Huayueyang, Beijing, China) and then reversed to cDNA with the TransScript II First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) [31 (link)]. The cDNAs were employed as templates to analyze the relative expression levels of several enzyme genes by qRT-PCR, and CpGAPDH was used as the internal gene [31 (link)]. All qRT-PCR reactions were performed using SYBR Green Master Mix (TaKaRa, Japan) on a Roche lightcycle96 real-time machine with the following programs: 95 °C for 30 s, then 40 cycles of 95 °C for 10 s and 60 °C for 20 s. Each qRT-PCR experiment had three biological and three technical replicates [31 (link)]. All of the primers are listed in Table S1 .
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!