m6A methyltransferase METTL3 expression in brain tissues of the NTD and control groups was detected by real-time qRT-PCR. Total RNA was extracted from brain tissues using TRIzol reagent (Invitrogen). Reverse transcription for cDNA synthesis was performed using the PrimeScript™ first strand cDNA Synthesis Kit (Takara, Beijing, China). Real-time qRT-PCR was conducted using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and the 7900HT Fast qPCR System (Applied Biosystems, Foster City, CA, USA). Cycling conditions were as follows: initial denaturation at 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 60 s, followed by a melt curve analysis from 60°C to 95.0°C in increments of 0.5°C per 10 s. The internal control was glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the relative expression levels of lncRNAs and genes were calculated using the 2−ΔΔCT method.
7900ht fast qpcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7900HT Fast QPCR System is a real-time PCR instrument designed for fast and accurate quantitative analysis of DNA and RNA samples. It utilizes microfluidic technology to enable rapid thermal cycling and data collection, providing efficient and high-throughput gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sds 2, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (5 mentions)
Micro amp 96, by Thermo Fisher Scientific (2 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Primescript first strand cdna synthesis kit, by Takara Bio (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Taqman fast universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Sybr green select master mix, by Thermo Fisher Scientific (1 mentions)
High capacity reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Sds software, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Pcr master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Qiashredder, by Qiagen (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Q pcr core kit master mix, by Eurogentec (1 mentions)
Sds v2, by Thermo Fisher Scientific (1 mentions)
12 protocols using 7900ht fast qpcr system
1
Quantifying METTL3 Expression in NTD
2
Quantitative Assessment of Neuronal Markers
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Quantitative PCR was performed using TaqMan® Gene Expression Assays with FAM reporter dye in TaqMan® Universal PCR Master Mix with UNG according to the manufacturer's instructions. Quantitative PCR reactions were carried out on Micro-Amp™ 96-well optical microtitre plates on the 7900HT Fast QPCR System (Applied Biosystems) using standard settings. TaqMan® Gene Expression Assays were used for NRGN (Hs00382922_m1), GAP-43 (Hs00967138_m1), SNAP-25 (Hs00938962_m1) and SYT-1 (Hs00194572_m1) (all from Applied Biosystems). Each reaction mixture consisted of 2.5 ng of cDNA and was run in duplicates. Quantitative PCR results were analyzed using the SDS 2.3 software (Applied Biosystems). The relative quantities were calculated following the ΔΔCT method (Livak and Schmittgen, 2001 ). Average CT:s of RPL-27 (Hs03044961_g1), RPL-30 (Hs00265497_m1) and HPRT-1 (Hs02800695_m1) were used as endogenous controls. All samples were normalized to the endogenous controls; the sample with the highest ratio was determined in each run and all the other samples were then compared to that sample (which was set to 1).
3
Quantitative Analysis of p53 and p21 Gene Expression
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TaqMan® Gene Expression Assays for p53, p21 and POLR2a (Pol II) were purchased from Applied Biosystems (Table 2 ). All primer products span exon–exon junctions to avoid amplification of genomic DNA. QPCR reactions were performed using 5 μl of cDNA in MicroAmp™ 96-well optical microtiter plates on a 7900HT Fast QPCR System in TaqMan® Fast Universal PCR Master Mix (Applied Biosystems) according to protocol (total volume 25 μl). cDNA was diluted 10 times prior to qPCR and all samples were run in duplicate. PCR results were analyzed with SDS 2.3 software (Applied Biosystems) and relative quantity was determined using the ΔΔCT Method [10 (link)], with DMSO-treated cells as calibrator and POLR2A as endogenous reference.
4
Quantitative RT-PCR Analysis of Stem Cell Differentiation
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Total RNA was isolated from samples using Trizol reagent (Life Technologies) and purification was performed following the manufacturers’ instructions. Reverse transcription was performed on 2 μg of RNA using Superscript reverse transcriptase III and random hexamer primers. TaqMan Universal PCR Master Mix was employed, and qPCR reactions were carried out on Micro-Amp 96-well optical microtiter plates with 7900 HT Fast QPCR System (Applied Biosystems). cDNA (2.5 ng) was used in the PCR and all samples were run in duplicate. The comparative threshold cycle values were normalized for the HPRT1 reference gene and the results were expressed as CT relative quantification using the ΔΔCT method. TaqMan gene expression assays for the following genes were carried out: NANOG (Hs02387400_g1); POU class 5 homeobox 1 (POU5F1/OCT4, Hs04260367_gH); paired box 6 (PAX6, Hs01088114_m1); LIN28 (Hs00702808_s1); alpha-fetoprotein (AFP, Hs01040598_m1); smooth muscle actin (SMA, Hs00426835_g1); β3-tubulin (TUJ1, Hs00801390_s1); SOX1 (Hs01057642_s1); microtubule associated protein 2 (MAP2, Hs00258900_m1); BCL2L11 (BIM, Hs00708019_s1); factor related apoptosis ligand (FASL, Hs00181226_g1); p53 upregulated modulator of apoptosis (PUMA, Hs00248075_m1); SOD2 (MnSOD, Hs00167309_m1); SYP (Hs00300531_m1); hypoxanthine phosphoribosyl transferase 1 (HPRT1, Hs02800695_m1).
5
Murine Skin Total RNA Extraction
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Total RNA from the full-thickness murine skin was extracted using a miRNeasy mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. cDNA was synthesized from RNA using a GoScript Reverse Transcription System (Promega, Madison, WI, United States). qPCR was performed in 96-well plates using a 7900HT Fast qPCR system (Applied Biosystems, Foster City, CA, United States); GAPDH expression was used as the internal control to normalize the expression of the target genes; fold change in expression was calculated using the ΔΔCt method. Primer sequences used to amplify the murine genes are listed in Supplementary Table S1 .
6
SOCS3 Expression in T Cell Subsets
7
Quantitative PCR Analysis of Cytoskeletal Proteins
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Quantitative PCR (qPCR) was performed using inventoried TaqMan Gene Expression Assays with FAM reporter dye in TaqMan Universal PCR Master Mix with UNG according to manufacturer's protocol, in a total reaction volume of 25 μL. qPCRs were carried out on Micro-Amp 96-well optical microtiter plates on a 7900HT Fast QPCR System (Applied Biosyste ms), using standard settings for Standard Curve qPCR. TaqMan Gene Expression Assays for the following genes were used: calpain-1 (Hs00559804_m1), calpain-2 (Hs00965097_m1), MAPT (microtubule associated protein tau) (Hs00902194_m1) and as the reference gene POLR2a (Hs00172187_m1). All samples were run in duplicates. PCR results were then analyzed with the SDS 2.3 software (Applied Biosystems) and the relative quantity of gene expression was determined using the ∆∆CT method [36] , with scrambled transfected cells as the calibrator and CT:s of POLR2a as the endogenous reference.
8
Time-Course Optimization and Viral Replication
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For time-course optimization (Figure S2 ), 10 h and 24 h replication experiments (Figure 7 ) Infected Vero-E6 cells and correspondent supernatants (150 μl), cultured in 6-well plates (2 ml culture medium per well - 3 independent wells per condition) were harvested at indicated time points and lysed in 500 μl of mastermix QVL lysis buffer from E.Z.N.A. Viral RNA kit used for Viral RNA extraction according to manufacturer’s instructions. RNA concentration was measured and 500 ng or 1000 ng of total RNA was used for cDNA synthesis using iScript. A 1:5 dilution of cDNA was used to perform quantitative real-time PCR (QPCR) using Applied Biosystems SYBR Green Master Mix on 7900 HT Fast QPCR System (Applied Biosystems) with SDS 2.4 Software. Primers used are described in Table S2 . All data (always in triplicate) were normalized to Ct values from three housekeeping (HK) genes ALAS-1, Guss and TBP, except for supernatants from replication experiments at 24 h, which Ct data were normalized using Ct values from correspondent cellular HK genes. Results were expressed as 2ˆ(-ΔΔCt)∗100%.
9
Quantitative PCR Analysis of Gene Expression
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Quantitative PCR was performed using inventoried TaqMan Gene Expression Assays with FAM reporter dye in TaqMan Universal PCR Master Mix with UNG according to manufacturer’s instructions, in a total reaction volume of 25 µL. qPCR reactions were performed on Micro-Amp 96-well optical microtiter plates on a 7900HT Fast QPCR System (Thermo Fisher Scientific), using standard settings for Standard Curve qPCR. TaqMan Gene Expression Assays (all from Thermo Fisher Scientific) are listed in Supplementary Table 2. 2.5 ng of cDNA was used in the PCR and all samples were run in duplicates. PCR results were analysed with the SDS 2.3 software (Applied Biosystems) and the relative quantity of gene expression was determined using the ∆∆CT method (Livak and Schmittgen 2001 (link)), with NPCs as the calibrator and average CT:s of RPL27 and HPRT1 as endogenous reference.
10
Quantitative PCR Analysis of Gene Expression
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Quantitative PCR was performed using inventoried TaqMan Gene Expression Assays with FAM reporter dye in TaqMan Universal PCR Master Mix with UNG according to manufacturer’s instructions, in a total reaction volume of 25 µL. qPCR reactions were performed on Micro-Amp 96-well optical microtiter plates on a 7900HT Fast QPCR System (Thermo Fisher Scientific), using standard settings for Standard Curve qPCR. TaqMan Gene Expression Assays (all from Thermo Fisher Scientific) are listed in Supplementary 2 . 2.5 ng cDNA was used in the PCR and all samples were run in duplicates. PCR results were analyzed with the SDS 2.3 software (Applied Biosystems) and the relative quantity of gene expression was determined using the ∆∆CT method65 (link), with BrainPhys-cultured cells as the calibrator and average CT:s of RPL27, RPL30 and HPRT1 as endogenous reference.
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