Total RNA was isolated with the ReliaPrepTM RNA Cell Miniprep System (Promega Z6010) and was reverse transcribed with Reverse Transcription System (Promega A3500). Real-time qPCR was performed with PikoReal 96 (Thermo Scientific). In each experiment, samples were run in triplicate and were normalized to β-actin to determine relative expression levels. Primer sequences were listed in Supplementary Methods.
Reliapreptm rna cell miniprep system
Manufactured by Promega
Sourced in United States, United Kingdom
The ReliaPrepTM RNA Cell Miniprep System is a lab equipment product designed for the rapid isolation and purification of total RNA from cultured cells. The system utilizes a silica-based membrane technology to efficiently capture and elute RNA, providing a streamlined workflow for RNA extraction.
Automatically generated - may contain errors
Lab products found in correlation
Goscripttm reverse transcription system, by Promega (4 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Gotaq qpcr master mix, by Promega (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Pikoreal96, by Thermo Fisher Scientific (1 mentions)
Reverse transcription system, by Promega (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Sybr green 1 master mix, by Roche (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Cfx connecttm real time pcr system, by Bio-Rad (1 mentions)
Pcr array data analysis web portal, by Qiagen (1 mentions)
31 protocols using reliapreptm rna cell miniprep system
1
Quantitative RNA Expression Analysis
2
Quantitative RT-PCR Analysis of Differentiating Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proliferating and 4-day differentiating cells’ pellets were prepared as detailed above. Total RNA was extracted using ReliaPrepTM RNA Cell MiniPrep System (Promega, Charbonnières-les-Bains, France) following manufacturer’s instructions and quantified at the Nanodrop. cDNA was prepared from 500 μg of total RNA using random hexamer and SuperScript III (Life technologies). Two μg of cDNA, 0.18 μL of each primers at 20 µM and 4.5 µl of SYBR-Green I Master mix (Roche, Meylan, France) were used per quantitative-RT-PCR assay and performed in triplicates using Roche LightCycler 480 II (Roche, Meylan, France). The primer list is provided in Table 2 .
3
Quantifying Neprilysin and MRPL49 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from SH-SY5Y cells using the ReliaPrepTM RNA cell miniprep system (Z6011, Promega, Milan, Italy) following the manufacturer’s instructions. Two micrograms of DNA-free total RNA were reverse transcribed into first-strand cDNA with random primers in a 20 μl final volume using the GoScriptTM reverse transcription system (Promega, A5000). The qPCR analysis was performed in technical triplicate using 40 ng of cDNA in 25 μl/well in 96-well plates, using the GoTaq® qPCR master mix (Promega, A6001). The CFX-ConnectTM Real-Time PCR system (Bio-Rad) was used. Beta-actin (ACTB) was quantified as reference housekeeping gene. The amplification steps were set as follows: a first step at 95°C for 10 min, 40 cycles (95°C for 15 s, 60°C for 1 min) and a final dissociation step (95°C for 15 s, 60°C for 20 s, 95°C for 15 s). The relative expression levels of neprilysin and MRPL49 transcripts were calculated using the ΔΔCt method. Statistical significance was assessed by either paired two-tailed t-test or two-way ANOVA. Primers design was performed by using the “Pick primers” tool of NCBI Nucleotide and manually adjusted to avoid the amplification of undesired sequences and to have comparable melting temperatures and reaction efficiency. Primer pairs sequences:
4
Comprehensive Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For gene expression analysis, ‘Human Adherens Junctions’ and ‘Human Extracellular Matrix and Adhesion Molecules’ RT2 Profiler PCR Arrays (Qiagen) were used. These RT2 Arrays profile simultaneously the expression of 84 genes involved in cell-cell contact and cell adhesion, within one sample. In brief, cells were harvested and mRNA was isolated using the ReliaPrepTM RNA Cell Miniprep System (Promega). The mRNA concentration was measured with a NanoDrop Fluorospectrometer (Thermo Fisher Scientific, Inc.; Waltham, MA, USA). For cDNA synthesis the RT2 First Strand Kit was used. A total of 0.5 µg RNA was reverse transcribed and the cDNA was amplified by the RT2 SYBR Green Master mix. Data were evaluated using the Qiagen PCR Array Data Analysis Web portal. Gene expression was normalized to the mean expression of the reference genes (GAPDH, ACTB, B2M HPRT and RPLP0) and was calculated using the ∆∆CT method.
5
RNA Extraction from Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular RNA was extracted at indicated time-points by the ReliaPrepTM RNA cell miniprep system (#Z6011, Promega, Madison, WI, USA) based on lysis with 1-thioglycerol as instructed by the manufacturer.
6
Quantitative Assessment of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted using a ReliaPrepTM RNA cell Miniprep system (Promega, Madison, WI, USA). The extracted total RNA was reverse-transcribed into cDNA using the ReverTra Ace® qPCR RT Master Mix with a gDNA remover kit (TOYOBO, Osaka, Japan). Three biological replicates were examined to ensure reproducibility. The RNA concentrations used for cDNA synthesis were 527.0, 629.4, and 96.3 ng/μL in the control groups and 524.8, 750.7, and 92.5 ng/μL in the UVA irradiation groups, respectively. IGFBP7 and Fos expression was measured using RT-qPCR with the following reaction mixture: 12.5 μL of TB Green® Premix Ex Taq II (Takara, Shiga, Japan), 2 μL of cDNA, 1 μL of 10 μM 1:1 forward and reverse target primers, and 8.5 μL of nuclease-free water. Using a Thermal Cycler Dice® Real Time System III (Takara, Shiga, Japan), the thermal cycling conditions were an initial denaturation step at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. The primers for IGFBP7, Fos, and GAPDH are listed in Table 1 . A single peak was observed for all amplicons in melt curve analysis. Gene expression was normalized to the geometric mean of GAPDH as an internal control, which was used as a representative housekeeping gene along with ACTB and 18S ribosomal RNA [23 (link)].
7
Sendai Virus Transduction PCR Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA from iPSCs and fibroblasts at day-7 of SeV transduction used as positive Sendai control was extracted using the ReliaPrepTM RNA Cell Miniprep System (Promega, Z6010) including DNase I treatment according to the manufacturer’s instructions. cDNA was generated from 1 µg of RNA using the GoScriptTM Reverse Transcription System (Promega, A5000) according to the manufacturer’s instructions.
For the PCR reaction mixture, 1 µl of cDNA produced from 1 µg of RNA was amplified using 10 µM dNTP mix, 5X Green GoTaq® Reaction Buffer and GoTaq® DNA Polymerase (5 u/µl) (Promega, M3175) and the following primers (10 µM) SeV-OCT4 forward: 5′-CCCGAAAGAGAAAGCGAACCAG-3′;SeV-OCT4 reverse: 5′-AATGTATCGAAGGTGCTCAA-3′; SeV-SOX2 forward: 5′-ATGCACCGCTACGACGTGAGCGC-3′; SeV-SOX2 reverse: 5′-AATGTATCGAAGGTGCTCAA-3′; SeV-Klf4 forward: 5′-TTCCTGCATGCCAGAGGAGCCC-3′; SeV-Klf4 reverse: 5′-AATGTATCGAAGGTGCTCAA-3′; SeV-cMYC forward: 5′-TAACTGACTAGCAGGCTTGTCG-3′; SeV-cMYC reverse: 5′-TCCACATACAGTCCTGGATGATGATG-3′; SeV forward: 5′-GGATCACTAGGTGATATCGAGC-3′; SeV reverse: 5′-ACCAGACAAGAGTTTAAGAGATATGTATC-3′. Oligonucleotides for the housekeeping gene- GAPDH were used as a positive control of the amplification reaction.
For the PCR reaction mixture, 1 µl of cDNA produced from 1 µg of RNA was amplified using 10 µM dNTP mix, 5X Green GoTaq® Reaction Buffer and GoTaq® DNA Polymerase (5 u/µl) (Promega, M3175) and the following primers (10 µM) SeV-OCT4 forward: 5′-CCCGAAAGAGAAAGCGAACCAG-3′;SeV-OCT4 reverse: 5′-AATGTATCGAAGGTGCTCAA-3′; SeV-SOX2 forward: 5′-ATGCACCGCTACGACGTGAGCGC-3′; SeV-SOX2 reverse: 5′-AATGTATCGAAGGTGCTCAA-3′; SeV-Klf4 forward: 5′-TTCCTGCATGCCAGAGGAGCCC-3′; SeV-Klf4 reverse: 5′-AATGTATCGAAGGTGCTCAA-3′; SeV-cMYC forward: 5′-TAACTGACTAGCAGGCTTGTCG-3′; SeV-cMYC reverse: 5′-TCCACATACAGTCCTGGATGATGATG-3′; SeV forward: 5′-GGATCACTAGGTGATATCGAGC-3′; SeV reverse: 5′-ACCAGACAAGAGTTTAAGAGATATGTATC-3′. Oligonucleotides for the housekeeping gene- GAPDH were used as a positive control of the amplification reaction.
8
RNA Extraction from Cell Pellets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell pellets (from gel disaggregation or cell trypsinisation) underwent RNA extraction with DNase digestion using ReliaprepTM RNA Cell Miniprep System (Promega). RNA quantitation was performed using the NanoDrop Spectrophotometer (Thermo Fisher Scientific).
9
Quantitative RT-PCR analysis of cytokine expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from CT-26 cells was extracted by using ReliaPrepTM RNA Cell Miniprep System, and cDNA was synthesized by using GoScriptTM Reverse Transcription System (both from Promega, Fitchburg, WI). Quantitative Real-time PCR was performed with SYBR Green PCR Master Mix (Applied Biosystems, CA). The following primer sets were used: CCL2, 5′-GCTGGAGCATCCACGTGTT-3′ and 5′-ATCTTGCTGGTGA ATGAGTAGCA-3′; CXCL2, 5′-CGCCCAGACAGAAGTCATAG-3′ and 5′-TCCTCCTTTCCAGGTCAGTTA-3′; CCL5, 5′-CACCA CTCCCTGCTGCTT-3′ and 5′-ACACTTGGCGGTTCCTTC-3′; CXCL9, 5′-TTTTGGGCATCATCTTCCTGG-3′ and 5′-GAGGTC TTTGAGGGATTTGTAGTGG-3′; CXCL10, 5′-CTTCTGAAAGG TGACCAGCC-3′ and 5′-GTCGCACCTCCACATAGCTT-3′; CCL11, 5′-GGCTGACCTCAAACTCACAGAAA-3′ and 5′-ACA TTCTGGCTTGGCATGGT-3′; LIF, 5′-ATGTGCGCCTAACATG ACAG-3′ and 5′-TATGCGACCATCCGATACAG-3′; L32, 5′-GAAACTGGCGGAAACCCA-3′ and 5′-GGATCTGGCCCTTGA ACC-3′.
10
qRT-PCR Analysis of Cytokine mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of total RNA was performed from cell pellets using ReliaPrepTM RNA cell Miniprep system (Promega, corporation, WI, USA) followed by cDNA synthesis using Goscript Reverse Transcription system (# A5001, Promega Corporation, WI, USA). QuantiTect SYBR Green PCR kit (Qiagen) was used for analysis of mRNA expression of each gene. The sequence of the primer used were: Il12a, 5′-CCACCC TT
GCCCTCCTAAAC-3′ and 5′-GGCAGCTCCCTCTTGTTGTG-3′; Il27, 5′-CTCTGCTTCCTC
GCTACCAC-3′ and 5′-GGGGCAGCTTCTTTTCTTCT-3′; Il23, 5′-AAGTTCTCTCCTCTT
CCCTGTCGC-3′ and 5′-TCTTGTGGAGCAGCAGATGTGAG-3′;Il6,5′ TATGAAGTTCCTCTCTGCAAGAGA-3′ and 5′- TAGGGAAGGCCGTGGTT-3′; Il1b, 5′-AAGGAGAACCAAGCAACGACAAAA-3′ and 5′-TGGGGAACTCTGCAGACTCAAACT 3′; Il10, 5′-ATAACTGCACCCACTTCCCAGTC-3′ and 5′CCCAAGTAACCCTTAAAGTCCTGC-3′;β-actin, 5′-TGTCCACCTTCCAGCAGATGT-3′ and 5′AGCTCAGTAACAGTCCGCCTAGA.
GCCCTCCTAAAC-3′ and 5′-GGCAGCTCCCTCTTGTTGTG-3′; Il27, 5′-CTCTGCTTCCTC
GCTACCAC-3′ and 5′-GGGGCAGCTTCTTTTCTTCT-3′; Il23, 5′-AAGTTCTCTCCTCTT
CCCTGTCGC-3′ and 5′-TCTTGTGGAGCAGCAGATGTGAG-3′;Il6,5′ TATGAAGTTCCTCTCTGCAAGAGA-3′ and 5′- TAGGGAAGGCCGTGGTT-3′; Il1b, 5′-AAGGAGAACCAAGCAACGACAAAA-3′ and 5′-TGGGGAACTCTGCAGACTCAAACT 3′; Il10, 5′-ATAACTGCACCCACTTCCCAGTC-3′ and 5′CCCAAGTAACCCTTAAAGTCCTGC-3′;β-actin, 5′-TGTCCACCTTCCAGCAGATGT-3′ and 5′AGCTCAGTAACAGTCCGCCTAGA.
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!