Total RNA was isolated from cells or tissues using TRIzol™ reagent (15596026, Thermo Fisher Scientific) according to the manufacturer's protocol. The total RNA concentration was measured using a Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific). Additionally, 1-μg of total RNA was used to synthesize cDNA with the iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA), as per the manufacturer's protocol. The generated cDNA was used as a template to perform real-time qPCR with a quantitative SYBR Green PCR mix on a QuantStudio 6 Flex Real-Time PCR system (Bio-Rad). Additionally, the relative expression of an individual gene was calculated using the 2−∆∆CT method, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control.
Quantstudio 6 flex real time pcr system
Manufactured by Bio-Rad
Sourced in United States
The QuantStudio 6 Flex Real-Time PCR System is a high-performance PCR instrument designed for accurate and precise quantification of nucleic acid samples. It features a 96-well block format and supports a wide range of sample types and applications, including gene expression analysis, microRNA profiling, and SNP genotyping.
Automatically generated - may contain errors
Lab products found in correlation
Itaq universal sybr green supermix, by Bio-Rad (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Iscript reverse transcription supermix, by Bio-Rad (1 mentions)
Hiscript 2 reverse transcriptase, by Vazyme (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Ssoadvanced universal sybr green master mix, by Bio-Rad (1 mentions)
Aurum total rna mini kit, by Bio-Rad (1 mentions)
Iscript reverse transcription, by Bio-Rad (1 mentions)
Illustra rnaspin mini rna isolation kit, by GE Healthcare (1 mentions)
Magmax 96 total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
7 protocols using quantstudio 6 flex real time pcr system
1
Quantitative Real-Time PCR: RNA Extraction and Analysis
2
Reverse Transcription and qPCR Analysis
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RNA was reverse transcribed to cDNA by using the HiScript II Reverse Transcriptase (Vazyme). After that, obtained cDNA was amplified on a QuantStudio 6 Flex Real-Time PCR System (Bio-Rad) through the standard procedures. All samples were amplified with forward and reverse primers. GAPDH was selected as internal control, and the results were represented as fold changes compared with controls.
3
Quantifying Cardiomyocyte Gene Expression
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Total RNA was isolated from cardiomyocytes cultured in 6-well plates using Trizol (Life Technologies, 15596018). Isolated total RNA was quantified, and normalized amounts were used as template to produce cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher, 11754050). Relative abundance of cDNA was assessed by qPCR on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR System using iTaq Universal SYBR Green Supermix (Bio-Rad, 1725124) and analyzed by using the delta-delta CT method. The following primers were used: Cdkn1a fw: 5′-GGGATGCATCTATCTTGTGATATG-3′, Cdkn1a rev: 5′-GTGGAACAGGTCGGACATCA-3′, Cyclin A fw: 5′-GGAAATTGCAGCTTGTCGGG-3′, Cyclin A rev: 5′-GCTGTCGCTTTGTGTACGTG-3′, Cyclin B fw: 5′-CAGACGATGGTGGTGATCCAA-3′, Cyclin B rev: 5′-TCCAGTGACTTCACGACCCA-3′, and Ppia fw: 5′-CTGAGCACTGGGGAGAAAGG-3′, Ppia rev: 5′-CACCCTGGCACATGAATCCT-3’. Gene expressions were normalized to Ppia expression.
4
Quantification of V. cholerae intIA Transcription
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Total RNA was prepared from co-incubation assays of V. cholerae with T. pyriformis and A. castellanii in 24-well plates. Bacterial cells from within protozoa were collected as described above and RNA was extracted by lysozyme digestion followed by use of the Aurum Total RNA mini kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The concentration of RNA was measured by spectrophotometry (NanoDrop ND-1000; NanoDrop Technologies). Complementary DNA (cDNA) was prepared from 400 ng RNA from each sample by iScript Reverse Transcription (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. Quantitative reverse-transcriptase PCR (qRT-PCR) was conducted using SsoAdvanced Universal SYBR Green Master Mix (Bio-Rad, Hercules, CA, USA) and a QuantStudio 6 Flex Real-Time PCR System using V. cholerae integron-integrase specific primers (intIA-1F/intIA-1R) (Supplementary Table 2 ). Transcription of intIA was determined relative to the transcription of gyrA (gyrA_F/gyrA_R) (Supplementary Table 2 ) using the comparative Ct (ΔΔCt) method.
5
Quantifying Cell Type Markers and SARS-CoV-2 Expression
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RNA was extracted to measure the expression of cell type marker genes (ciliated cell: DNAH1, DNAH5, FOXJ1, and TEKTIN; secretory cell: MUC5AC, MUC5B, and TFF3; basal cell: ITGA6 and KRT5), SARS-CoV-2 receptors, and SARS-CoV-2. Total RNA from SARS-CoV-2 infected cells was extracted using MagMAX™-96 Total RNA Isolation Kit (Invitrogen) or TRIzol (Invitrogen). Total RNA from all the other experiments was extracted using illustra RNAspin Mini RNA Isolation Kit (GE Healthcare). RNA extracts were reverse transcribed using a High-Capacity RNA-to-cDNA Kit (Applied Biosystem). Real-time PCR was performed on QuantStudio 6 Flex Real-Time PCR System using iTaq® Universal SYBR® green supermix (Bio-Rad). 18S ribosomal RNA was used as reference. Primer sequences are documented in Supplementary Table S5 .
6
Quantitative PCR for Gene Expression Analysis
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Total RNA was isolated from cells using the TRIzol reagent (Life Technologies, Grand Island, NY, USA), and 0.5 μg of total RNA was reverse transcribed to generate cDNA using the iScript cDNA Synthesis Kit (1708891, Bio‐Rad) according to the manufacturer's instructions. qPCRs were performed using the iTaq Universal SYBR Green supermix (1725121, Bio‐Rad) on the ABI QuantStudio 6 Flex Real‐Time PCR System. ACTB expression was used for normalization. Primers used in the study are listed in Table 1 .
7
Transcriptional Profiling of Mycobacterium tuberculosis
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Mycobacterium tuberculosis genes that were differentially expressed between the active stage and NRP stage, including narJ, tcrY, uvrC, Rv1356c and Rv3134c, were selected for further study. The primer sequences used in this study are listed in Table 1 . The expression of Mtb genes was examined by qRT-PCR. The PCR reaction was performed on a real-time PCR instrument (Applied Biosystems QuantStudio 6 Flex Real-Time PCR System) using SsoFast™ EvaGreen® Supermix (Bio-Rad Laboratory, Inc., USA). The relative expression of genes was examined by 2-ΔΔCt. The 16S rRNA gene was used as an internal control.Table 1
Primer pairs used to amplify cDNA.
| Gene | Direction/position of primer | Sequence |
|---|---|---|
| 16S rRNA | Forward, 469-491 | 5′-TTGACGGTAGGTGGAGAAGAAGC-3′ |
| Reverse, 909-888 | 5′-CCTTTGAGTTTTAGCCTTGCGG-3′ | |
| narJ | Forward, 439-512 | 5′-CCTATGAGTACACCGTGGCG-3′ |
| Reverse, 603-584 | 5′-GGGACGGTCAAGGTAAACGG-3′ | |
| tcrY | Forward, 796-815 | 5′-CATGAACTGCGAACTCCCCT-3′ |
| Reverse, 930-911 | 5′-GACGAGACGTGTTATCCGCT-3′ | |
| uvrC | Forward, 114-133 | 5′-CGAGTCATCTACGTCGGCAA-3′ |
| Reverse, 308-289 | 5′-GAATCGCGGATCGAACTCCT-3′ | |
| Rv1356c | Forward, 531-550 | 5′-CCTACCGCTAAGCATGTCCC-3′ |
| Reverse, 643-624 | 5′-CCGGGATTCTCGCTGCTATT-3′ | |
| Rv3134c | Forward, 472-491 | 5′-GAGGTGGACAATGGTGTGGT-3′ |
| Reverse, 610-591 | 5′-TTCGACGTCATCGGGTGTTT-3′ |
The 16S rRNA gene was used as a housekeeping gene to normalize the data. Primers from Haile et al. (2002) [13 (link)].
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