Total RNA was extracted from cells and reverse transcribed to cDNA using RNAiso plus (Invitrogen, Waltham, MA, United States) and 5 × PrimeScript RT Master Mix (Promega, Madison, WI, United States).
Primescript rt master mix
Manufactured by Promega
Sourced in United States
PrimeScript RT Master Mix is a ready-to-use solution for reverse transcription. It contains PrimeScript Reverse Transcriptase and necessary reagents for efficient cDNA synthesis from RNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Rnaiso plus, by Thermo Fisher Scientific (3 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 2 real time qrt pcr system, by Roche (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Roche (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Taq polymerase, by Takara Bio (1 mentions)
Ddh2o, by Takara Bio (1 mentions)
Sybr green mix, by Takara Bio (1 mentions)
Trizol reagent, by Absin (1 mentions)
Lightcycler 96 real time pcr system, by Roche (1 mentions)
Sybr green pcr kit, by Roche (1 mentions)
Power sybr green pcr master mix, by Takara Bio (1 mentions)
Prism 6, by GraphPad (1 mentions)
7 protocols using primescript rt master mix
1
RNA Extraction and cDNA Synthesis
2
Granulosa Cell RNA Extraction and qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from granulosa cells with TRIzol Reagent (Invitrogen); its concentration and purity were measured by NanoDrop 2000 (Thermo Fisher Scientific), and the reverse transcription was conducted by PrimeScript™ RT Master Mix (Promega). qRT-PCR was performed on LightCycler® 480 II real-time qRT-PCR system (Roche) with SYBR Green master mix (Takara). Sequences of Primers (Tsingke, Nanjing, China) used in the study were as follows: GAPDH: forward 5’-GGAGCGAGATCCCTCCAAAAT-3’; reverse 5’ –GGCTGTTGTCATACTTCTCATGG-3’. DAPK2: forward 5’-TGCAGCCAAGTTCATCAAGAAGCG-3’; reverse 5’-ACACTAGCTCAAGGATGAGCACCA-3’. Relative gene expression was calculated with the 2–ΔCT method.
3
Quantitative RT-PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the HeLa and SiHa cells using TRIzol (Invitrogen; Milan, Italy) according to manufacturer’s protocol, and 500 ng was used to obtain cDNA through reverse transcription using PrimeScript RTMasterMix (Promega; Madison, WI, United States). Quantitative RT-PCR was conducted using an SYBR Green PCR master mix (Roche; Basel, Switzerland) on the CFX96 Real-Time PCR Detection System (Bio-Rad; Hercules, CA, United States). PCR amplification was performed with the specific primer sets as described previously [12 (link)]. Relative expression was normalized to the expression of β-actin. The 2−ΔΔCt method was used for relative quantification of gene expression. The primers used in the studies were: PKM2 sense, 5′-ATGTCGAAGCCCCATAGTGAA-3′, PKM2 antisense, 5′-TGGGTGGTGAATCAATGTCCA-3′, β-actin sense, 5′-CTCCATCCTGGCCTCGCTGT-3′, β-actin antisense, 5′-GCTGTCACCTTCACCGTTCC-3′.
4
Quantifying Gene Expression in Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA of the lung tissue was obtained using TRIzol reagent. The reverse transcription reactions for mRNA were performed using PrimeScript™ RT Master mix (Promega Corporation, Madison, WI, USA). The RT-qPCR analysis was performed using Taq polymerase (Takara Biotechnology Co., Ltd., Dalian, China) consisting of a final volume of 20 µl, containing 2 µl cDNA, 10 µl SYBR-Green Mix, 4 µl primer mix and 4 µl ddH2O (Takara Biotechnology Co., Ltd.). Primers specific for the TNF-α, IL-6, IL-1β, glyco-protein-B (gB) gene and β-actin are listed in Table I . β-actin was selected as an internal control to normalize target gene expression. Thermocycling conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec, then a melting curve analysis from 60°C to 95°C every 0.2°C for 1.5 min was obtained. The transcript amount was normalized to U6 and β-actin and quantified using the 2−ΔΔCq method (17 (link)).
5
Quantitative Analysis of SGLT1 and NHE3 Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was extracted using RNAiso plus (Invitrogen, USA) reagent and subjected to reverse transcription with 5× PrimeScript RT Master Mix (Promega, USA). A quantitative real-time PCR (qPCR) analysis was performed to amplify the SGLT1 and NHE3 genes using the cDNA as the template and the β-actin gene as an internal standard. The qPCR reaction contained 10 μL of SYBR Premix ExTaq II (Takara Bio, China), 0.5 μL forward primer, 0.5 μL reverse primer, 1 μL of cDNA template, and RNase-free ddH2O was added to 20 μL. The reaction protocol was as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s; each sample was repeated three times. These primers are presented in Table 1. Data analysis was based on the measurement of the cycle threshold (Ct). The relative level of SGLT1 and NHE3 mRNA expression were calculated using the 2−△△Ct method.
6
RNA Extraction and Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRIzol reagent (Absin, Shanghai, China) was used to extract total RNA. Then the quantity and quality of the RNA was determined using a SpectraMax Plus 384 enzyme-labeling instrument. Reverse transcription reactions were performed using Prime Script RT Master Mix (Promega) according to the manufacturer’s instructions. In addition, real-time PCR was performed using a LightCycler® 96 Real-Time PCR System (Roche) and SYBR Green PCR kit (Roche). Gene expression was evaluated using the 2−ΔΔCt method and normalized to the housekeeping gene actin beta (ACTB). The sequences of the utilized PCR primers used are listed in Supplementary Table S1 and were purchased from Sangon Biotech (Shanghai).
7
PEDV M Gene Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect virus replication, the relationship between copy number (X) and cycle threshold (Ct) (Y) was established based on the amplification efficiency of the PEDV membrane (M) gene in the PCR instrument. The primers for PEDV M (Table 3 ) were used to quantify the number of copies of PEDV M. The PEDV M gene plasmid was preserved in our laboratory. The Power SYBR Green PCR Master Mix (Takara, Dalian, China) was used to carry out the PCR reactions according to the manufacturer’s instructions. We used GraphPad Prism 6 software (GraphPad Inc., La Jolla, CA, USA) to analyze the data based on the cycle δCt method. RNA from collected cell samples was extracted using RNAiso plus (Invitrogen, Waltham, MA, USA). Then, 5 × PrimeScript RT Master Mix (Promega, Madison, WI, USA) and the total viral RNA were used to generate cDNA. The M gene was amplified using quantitative real-time polymerase chain reaction (qPCR) in a reaction comprising 10 μL of SYBR PreMix ExTaq II (Takara), 0.5 μL of the forward primer, 0.5 μL of the reverse primer, 2 μL of the cDNA template, and 5 μL of H2O. The reaction was preprocessed for 30 s at 95 °C, followed by 40 cycles of 95 °C for 5 s and 64 °C for 30 s. For each sample, the process was repeated three times. Data analysis was based on the Ct measurements. The relative expression levels of PEDV M mRNA were then calculated.
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!