500 ng of RNA, obtained by RIP, were reverse transcribed to cDNA using random exaprimers and MMLV reverse transcriptase (Life Technologies, Carlsbad, CA, USA). Real-time PCR was performed using Platinum Sybr Green QPCR supermix (Life Technologies) on the ABI Prism 7300 Sequence Detection Systems (Applied Biosystems). MAD2 and MARCH3 oligonucleotide primers were purchased from Sigma-Aldrich and their sequences are available upon request.
Platinum sybr green qpcr supermix
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, China
The Platinum SYBR Green qPCR SuperMix is a ready-to-use solution for quantitative real-time PCR (qPCR) assays. It contains all the necessary components, including SYBR Green I dye, for the detection and quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (27 mentions)
Trizol, by Thermo Fisher Scientific (19 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (9 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (8 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (7 mentions)
Superscript 3 rt, by Thermo Fisher Scientific (6 mentions)
Takara pcr thermal cycler dice, by Takara Bio (6 mentions)
Rneasy kit, by Qiagen (5 mentions)
Random hexamer, by Thermo Fisher Scientific (5 mentions)
Mulv reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Qiagen (4 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (4 mentions)
Rneasy micro kit, by Qiagen (4 mentions)
Steponeplus 96 well real time pcr system, by Thermo Fisher Scientific (4 mentions)
Abi prism 7300 sequence detection system, by Thermo Fisher Scientific (3 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (3 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Superscript 2 reverse transcriptase kit, by Thermo Fisher Scientific (3 mentions)
108 protocols using platinum sybr green qpcr supermix
1
Real-Time PCR Analysis of MAD2 and MARCH3
2
qPCR Analysis of Immune Genes in DCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from 5,000 to 10,000 DC previously facs-sorted into 40 μl of lysis buffer (Life Technologies). Dynabeads mRNA Direct Purification Kit (Life Technologies) was used following manufacturer's guidelines. RNA was reverse transcribed with High Capacity cDNA Transcription Kit (Applied Biosystems). PCR were performed with Platinum SYBR Green qPCR SuperMix (Life Technologies) and QuantStudio 6 Flex (Applied Biosystems). Quantification of the PCR signals of each sample was performed by comparing the cycle threshold values (Ct), in duplicate, of the gene of interest with the Ct values of the TBP housekeeping gene.
Primers used in the research: Il6 Fwd 5′-CCTCTCTGCAAGAGACTTCCAT-3′, Il6 Rev 5′-ACAGGTCTGTTGGGAGTGGT-3′, Il12a (p35) Fwd 5'-GCCACCCTTGCCCTCCTAA-3', Il12a (p35) Rev 5'-GGTTTGGTCCCGTGTGATGTC-3', Irf1 Fwd 5′-GTTGTGCCATGAACTCCCTG-3′, Irf1 Rev 5'-TGGACTTTCTCTCTTTCCTCTGG-3′, Ccr7 Fwd 5′-CTCCTTGTCATTTTCCAGGTGTG-3′, Ccr7 Rev 5′-GGCAGGAACCAGGCCTTAAA-3′, Tbp 5′-GAAGCTGCGGTACAATTCCAG-3′, Tbp Rev 5'-CCCCTTGTACCCTTCACCAAT-3′.
Primers used in the research: Il6 Fwd 5′-CCTCTCTGCAAGAGACTTCCAT-3′, Il6 Rev 5′-ACAGGTCTGTTGGGAGTGGT-3′, Il12a (p35) Fwd 5'-GCCACCCTTGCCCTCCTAA-3', Il12a (p35) Rev 5'-GGTTTGGTCCCGTGTGATGTC-3', Irf1 Fwd 5′-GTTGTGCCATGAACTCCCTG-3′, Irf1 Rev 5'-TGGACTTTCTCTCTTTCCTCTGG-3′, Ccr7 Fwd 5′-CTCCTTGTCATTTTCCAGGTGTG-3′, Ccr7 Rev 5′-GGCAGGAACCAGGCCTTAAA-3′, Tbp 5′-GAAGCTGCGGTACAATTCCAG-3′, Tbp Rev 5'-CCCCTTGTACCCTTCACCAAT-3′.
3
Gene Expression Analysis of Differentiated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were differentiated for the respective days and sorted on a FACS Aria II. RNA was collected using RNA MiniPrep Plus (Invitrogen) and quantified on a NanoDrop (GE Healthcare). Equal amounts of RNA were used for cDNA synthesis using SuperScript III First-Strand Synthesis System (Life Technologies). qPCR was conducted using Platinum SYBR Green qPCR SuperMix (Life Technologies). The reactions were run on a Mastercycler RealPlex Thermal Cycler (Eppendorf) and the expression levels were calculated by minimal cycle threshold values (Ct) normalized to the reference expression of RPL13a. The qPCR products were run on an agarose gel and stained with ethidium bromide to confirm specificity of the primers. Primer sequences can be found in Supplementary Table 3 .
4
Quantification of mRNA Levels in Thyroid Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
150 ng of total RNA of Nthy-ori-3.1 and BCPAP cells, transfected with siRNA1 or control, were reverse transcribed to cDNA using random exaprimers and SuperScript III reverse transcriptase (Life Technologies, Carlsbad, CA, USA). Real-time PCR was performed using Platinum Sybr Green QPCR supermix (Life Technologies) with the ABI Prism 7300 Sequence Detection Systems (Applied Biosystems). The ΔΔCT method, by means of the SDS software (Applied Biosystems), was used to calculate mRNA levels. Oligonucleotide primers were purchased from Sigma-Aldrich and their sequences are available upon request.
5
RNA Isolation and qPCR Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sorted primary MECs were maintained at 4°C and collected by centrifugation. RNA was harvested with the Absolutely RNA Microprep Kit (Stratagene) according to the manufacturer's instructions, and reverse transcribed with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). The cDNA was amplified by Platinum SYBR Green qPCR Supermix (Life Technologies, Inc.) on a Rotor-gene 6000 real-time cycler (Corbett Research, Australia). The TATA-binding protein (TBP) was used as an internal control gene, and primers used were as described in [20 (link)].
6
Dlst Alternative Splicing Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA extraction from purified B cells was performed using TRIzol (LifeTech). 1 μg of RNA was treated with DNase I prior reverse transcription into cDNA. Analysis of Dlst alternative splicing events were performed by RT-PCR using specific primers (table S3 ). Intron retention was quantified by qPCR whereas alternative exon inclusion was quantified after gel densitometry of the PCR products. qPCR assays were performed using Platinum® SYBR® Green qPCR SuperMix (Life Technologies). mRNA expression of Dlst and Elavl genes was quantified by the ΔΔCT method (comparative threshold cycle) and normalised to the expression of 18S rRNA.
7
Quantitative Analysis of Cell Cycle Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from sorted plasmablast populations using TRIzol (LifeTech). RNA from sorted plasmablast B cells was converted to cDNA according to the superscript reverse transcriptase III protocol (Invitrogen) and then analysed by RT-qPCR. Cell cycle genes E2F1, E2F2, Myc, and Myb were analysed using custom or commercially available primers (see Table S3). E2F1 and E2F2 mRNA transcript expression was analysed using primers according to Pilon et al (2008) (link). Myc, E2F1, and E2F2 RT-qPCR assays were analysed using Platinum SYBR Green qPCR SuperMix (Life Technologies). Relative abundance was calculated using a standard curve or δ CT method and normalized to the expression of mRNA-encoding HPRT. Myb RT-qPCR assays were performed with Taqman assays. Expression of Myb mRNA was calculated using a standard curve and normalized to the expression of β2M.
Table S3
8
Quantitative RNA Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA extraction from purified cell lines was performed by using TRIzol (Life Technologies, Gaithersburg, MD, USA). 1 μg of RNA was treated with DNase I prior and then reverse transcription into cDNA. qRT-PCR assays functioned by using Platinum® SYBR® Green qPCR SuperMix (Life Technologies) which made housekeeping gene GAPDH or U6 the internal control. The 2−ΔΔCt (comparative threshold cycle) method was employed to identify the relative quantification of gene expression levels. RiboBio (Guangzhou, China) was employed to synthesize the premier sequences, and the results were shown in Table S1.
9
Validating RNAseq Data with qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNAseq results were validated by real-time PCR. Triplicate quantitative assays were performed using a Platinum SYBR Green qPCR SuperMix (Life Technologies, Carlsbad, CA, United States). Cells from control cultures (N2 sparging) were used as calibrators and RNA 16S served as endogenous reference gene (Okonkwo et al., 2017 (link)). Calculation of gene expression was carried out using the 2–ΔΔCt method as in Livak and Schmittgen (2001) (link). For each sample, mRNA amount was calculated relatively to the calibrator sample for the corresponding genes. Primers used for genes expression analysis are listed in Supplementary Table 2 .
10
Quantification of ZFP36 Family Transcripts in B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from purified B cells using TRIzol (LifeTech) or RNeasy Micro or Mini Kit (Qiagen). RNA was treated with DNase I before reverse transcription into cDNA. ZFP36 family expression was analysed using custom and commercially available TaqMan assays with specific primers (Supplementary Table 9 ). Expression of mRNA was calculated using a standard curve and normalised to the expression of β2M. Genomic DNA was extracted from purified B cells using Cell lysis solution (Qiagen) containing proteinase K (Roche). Protein was removed by salt precipitation, and the DNA was isolated using isopropanol. Relative abundance of ZFP36l1 exon 2 was analysed by quantitative PCR with specific primers (Supplementary Table 9 ), qPCR assays were performed with Platinum SYBR Green qPCR SuperMix (Life Technologies). Relative abundance of ZFP36l1 was calculated using the comparative threshold cycle (ΔΔCT) method and results were normalised to the expression of TBP.
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!