Total RNA was extracted from tissue or cell culture samples by using the Trizol reagent (#A33251, Invitrogen) according to the manufacturer’s instructions, and 1–2 μg of RNA was treated with RNase-free DNase (#AM1906, Invitrogen) to remove genomic DNA contamination. Oligonucleotide primer sequences used in the reverse transcriptase-PCR are listed in Supplementary Data 2 . qRT-PCR analysis was conducted with a SYBR Green Supermix kit (#4368706) with ABI7500 (both from Applied Biosystems, Foster City, CA, USA). The cycle parameters were 95 °C for a 1 min hot start and 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 20 s. The fold change in expression was calculated using the 2−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. Experiments for each sample were done in triplicate.
Sybr green supermix kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SYBR Green Supermix Kit is a ready-to-use solution containing SYBR Green I dye, DNA polymerase, dNTPs, and optimized buffer components for real-time PCR applications. The kit is designed to provide consistent and reliable detection and quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Rnase free dnase, by Thermo Fisher Scientific (2 mentions)
Abi 7500, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Promega (1 mentions)
One step rna pcr kit, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Roche (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
9 protocols using sybr green supermix kit
1
RNA Extraction and qRT-PCR Analysis
2
Quantitative RT-PCR Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from tissue or cell culture samples by using the Trizol reagent (#A33251, Invitrogen) according to the manufacturer's instructions, and 1-2μg of RNA was was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which this version posted July 7, 2020. ; https://doi.org/10.1101/2020.07.07.190496 doi: bioRxiv preprint treated with RNase-free DNase (#AM1906, Invitrogen) to remove genomic DNA contamination.
Oligonucleotide primer sequences used in the reverse transcriptase-PCR are listed in Supplementary Note 1. qRT-PCR analysis was conducted with a SYBR Green Supermix kit (#4368706) with ABI7500 (both from Applied Biosystems, Foster City, CA, USA). The cycle parameters were 95°C for a 1 min hot start and 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 20 s. The fold change in expression was calculated using the 2 -ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control.
Experiments for each sample were done in triplicate.
The copyright holder for this preprint (which this version posted July 7, 2020. ; https://doi.org/10.1101/2020.07.07.190496 doi: bioRxiv preprint treated with RNase-free DNase (#AM1906, Invitrogen) to remove genomic DNA contamination.
Oligonucleotide primer sequences used in the reverse transcriptase-PCR are listed in Supplementary Note 1. qRT-PCR analysis was conducted with a SYBR Green Supermix kit (#4368706) with ABI7500 (both from Applied Biosystems, Foster City, CA, USA). The cycle parameters were 95°C for a 1 min hot start and 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 20 s. The fold change in expression was calculated using the 2 -ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control.
Experiments for each sample were done in triplicate.
3
Quantifying BCL11A Expression via RT-qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The methods for isolating RNA was described in previous study.18 (link) We used a PrimeScriptRT Reagent Kit for reverse transcription following the manufacturer’s instructions (Promega, Madison, WI). We performed the Real-time PCR on a Bio-Rad CFX100 using a SYBR Green SuperMix kit (Invitrogen, Carlsbad, CA). We used β-actin as a control for each group. The BCL11A and β-actin primer sequences: BCL11A-F 5-CAGCACTTAAGCAAACGGGAAT-3 and BCL11A-R 5-TTGTTTCCGTTTGTGCTCGATA-3; β-actin-F 5-GGACTTCGAGCAAGAGATGG-3 β-actin-R 5-ATCTGCTGGAAGGTGGACAG-3. The primers were purchased from Invitrogen (Shanghai, People’s Republic of China).
4
Quantitative Analysis of CCRK and miR-335-5p
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the cells with TRIzol™ (Cat#15596018, Invitrogen, USA). Reverse transcription PCR was performed using a one-step RNA PCR kit (Cat#RR064B, TaKaRa, China). SYBR Green Supermix kit (C11733046, Invitrogen, USA) was used to perform real-time quantitative PCR (RT-qPCR) on an ABI PRISM® 7500 sequence detection system. The primer sequences used for RT-qPCR were synthetized by Saiyin Biotechnology (Shanghai) Co., Ltd., and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference. The sequences of the primers used for RT-qPCR were as follows: CCRK-Forward: 5′-CCTCCATCAGTACTTCTTCACA-3′; CCRK-Reverse: 5′-GAATCAGCTCTGGGTTCAAC-3′; miR-335-5p Forward: 5′-ACACTCCAGCTGGGTCAAGAGCAATAACGAAA-3′; miR-335-5p Reverse: 5′-CTCAACTGGTGTCGTGGA-3′; and miR-335-5p RT primer: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACATTTTTC-3′. Every experiment was repeated thrice.
5
Quantitative Analysis of HtrA1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using a Trizol Reagent (Thermo Fisher Scientific, USA) and cDNA was synthesized using a Revert Aid First Strand c DNA Synthesis Kit (Thermo Fisher Scientific, USA), according to the manufacturer’s protocol. Quantitative RT-PCR analyses were performed in technical triplicates using a SYBR Green Supermix kit (Thermo Fisher Scientific, USA). The expression levels of HtrA1 were determined using the 2–ΔΔCt method and normalized to the housekeeping genes β-actin or GAPDH. All primers sequences are shown in supplementary Table 3 .
6
Quantification of Thyroid Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRIzol reagent (Life Technologies, Carlsbad, California, U.S.A.) was used to extract total RNA of human primary thyrocytes. The RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). The primers for TPO, NIS, Pendrin, MCT8, CBS, CSE, 3-MPST, and GAPDH are listed in Table 1 . Detection of mRNA expression was carried out by the SYBR Green Supermix Kit (Thermo Fisher) and 7,500 real-time PCR system (Applied Biosystems, Foster City, CA, United States). Relative target gene expression was quantified by the 2−ΔΔCt method using GAPDH expression for normalization.
7
Quantitative Analysis of NTSR1 in Gastric Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated and purified from noncancerous gastric mucosal and gastric cancer tissues, as well as cancer cell lines, using the RNeasy kit (Qiagen, USA), and mRNAs were first reverse transcribed into cDNA using the cDNA synthesis kit (Roche, Germany). The qRT-PCR reactions were performed using a SYBR Green supermix kit (Thermo Scientific, USA). NTSR1 mRNA was normalized against mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Sequences of the primers were as follows: NTSR1: sense, 5′-CAGGTCAACACCTTCATGTCC-3′ and antisense, 5′-ATGCTG AATGTGCTGTGCTC-3′; and GAPDH: sense, 5′-AAATCAAGT GGGGCGATGCTG-3′ and antisense, 5′-GCAGAGATGATGA CCCTTTTG-3′. Data were reported as relative quantities according to an internal calibrator using the 2−ΔΔCT method (Pfaffl, 2001 (link)). All samples were measured in triplicate, and the mean values were used for quantification.
8
Quantifying SARS-CoV-2 Host Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from thyrocytes by TRIzol reagent (Life Technologies, Carlsbad, CA, USA). Then, the RNA was reverse-transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA). The primers for ACE2, NRP1, TMPRSS2, and GAPDH are presented in Table 2 . An SYBR Green Supermix Kit (Thermo Fisher, Woolston, UK) was used to determine mRNA expression. The relative expression of each gene was normalized to GAPDH expression.
9
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions, and the RNA concentration was determined using NanoDrop (Thermo Fisher Scientific) and reverse-transcribed into complementary DNA (cDNA) by the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). qRT-PCR was performed using the SYBR Green Supermix Kit (Thermo Fisher Scientific) in Bio-Rad PCR equipment. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control, and gene expression levels were determined using the 2−ΔΔCt method. All primer sequences are shown in table S2.
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!