The PCR protocols followed those described by Chen et al. [31 (link)], and utilized the same plasmid and reference chromosomal amplicons as well as the primer pairs described by them. The amplification reactions were carried out with ABI PRISM 7900HT SDS using the SYBR Green Master Mix (Applied Biosystems). The number of cycles required to reach the CT number (preset threshold) for each DNA sample was calculated from six separate experiments. The relative change in the copy number of a plasmid between two strains, normalized to the chromosomal reference sequence, was calculated by the 2-ΔΔCT analysis [47 (link)].
Abi prism 7900ht sd
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI PRISM 7900HT SDS is a real-time PCR instrument designed for high-throughput gene expression analysis. It utilizes the SDS (Sequence Detection System) technology to accurately quantify and analyze nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Gotaq qpcr master mix, by Promega (6 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
Nanodrop nd 100, by Thermo Fisher Scientific (5 mentions)
Taqman mgb probe, by Thermo Fisher Scientific (4 mentions)
7900ht fast real time pcr system instrument, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Sds 2.2.1 program, by Thermo Fisher Scientific (3 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Omniexpress chip, by Illumina (2 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Quantstudio real time pcr software, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
26 protocols using abi prism 7900ht sd
1
Quantitative Real-Time PCR Analysis
2
Quantitative PCR Analysis of Gene Expression
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cDNA was synthesized from total RNA using the QuantiTect reverse transcription kit (Qiagen, Hilden, Germany). Quantitative PCR (QPCR) was performed by using ABI Prism 7900HT SDS and SYBR green chemistry (Applied Biosystems, Carlsbad, CA) and primers as indicated in Table S1 . The primer pairs had an amplification efficiency of 91.5% −112.5%. Expression of genes was related to levels of actin mRNA using the 2-ΔΔCT method using QuantStudio Real-Time PCR Software (Applied Biosystems, Carlsbad, CA), setting the relative expression of the F12 sample at 1. Data were analyzed by t test comparing F12 and B12 samples after 12 and 48 h.
3
Quantifying Liver RNA and Gene Expression
4
Quantitative Analysis of ASFV Gene Expression
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The 6·106 PAMs were seeded on p60 plates and infected with the indicated viral strains. At 16 hpi, RNA was extracted from the cells using the RNeasy kit (Qiagen, Venlo, The Netherlands), and total RNA was quantified with the NanoDrop One (Thermo Scientific). Equivalent amounts of RNA were then retrotranscribed using the NZY first-strand cDNA kit (NZYTech), and RT-PCR was performed in technical triplicate with a cDNA amount of 12 ng/sample on ABI PRISM 7900HT SDS (Applied Biosystems, Waltham, MS, USA) thermal cycler using SYBR Green (NZYTech). Expression levels of genes of interest were normalized against 18s ribosomal RNA (rRNA) expression, and these values were relativized against the mean of the values obtained in the mock. The primers used are as follows: 18S: 5′-GGCCCGAGGTTATCTAGAGTC-3′, 5′-TCAAAACCAACCCGGTCA-3′; CP204L: 5′-AAAA ATGATAATGAAACCAATGAATG-3′, 5′-ATGAGGGCTCTTGCTCAAAC-3′; MGF505-2R: 5′-GAGTCCACCTTGGTGATAAAG-3′, 5′-CCATGATCGTCCTCACTTTC-3′; IFN-β: 5′-GTGGAACTTGATGGGCAGAT-3′, 5′-TTCCTCCTCCATGATTTCCTC-3′.
Animal Experiment I was performed at the Korea Zoonosis Research Institute, Jeonbuk National University, Republic of Korea.
Animal Experiment I was performed at the Korea Zoonosis Research Institute, Jeonbuk National University, Republic of Korea.
5
Hippocampal RNA Extraction and qPCR Analysis
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Cultured hippocampal neurons or mice hippocampi were homogenized in Trizol Reagent (Life Technologies ref.: 15596018) and the RNA was extracted using Direct-zolTM RNA minipreps (Zimo research ref.: R2052). RNA was quantified at 260 nm absorbance using a Nanodrop ND-100 (Themo Fisher Scientific). First strand cDNA was obtained using RevertAid H Minus First Strand cDNA Synthesis kit (Themo Fisher Scientific ref.: K1631). 5 ng of synthesized cDNA were used to perform the qPCR using GoTaq® qPCR Master Mix (Promega ref.: A6002) in ABI PRISM 7900HT SDS (Applied Biosystems; Life Technologies). Primers obtained from Sigma-Aldrich were used at 0.5 μM final concentration (see list below). Three housekeeping genes Gapdh, Gus-B and Pgk-1 were used as endogenous controls.
6
Quantifying NSM2 Transcripts in Brain Cortex
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Total RNA from brain cortex homogenates was obtained by Trizol Reagent (Ambion /RNA. Life Technologies Co., Grand Island, NY, USA) and chloroform extraction. RNA was further cleaned up using Rneasy Mini kit (Qiagen, Hilden, Germany). RNA concentration was estimated by absorbance at 260 nm using a Nanodrop ND-100 (Thermoscientific; Themo Fisher Scientific Inc.). The retrotranscription to first strand cDNA war performed using RevertAid H Minus First Strand c DNA Synthesis kit from Thermo Scientific. qPCR was performed using GoTaq® qPCR Master Mix (Promega Co., Madison, WI, USA) and ABI PRISM 7900HT SDS (Applied Biosystems; Life Technologies Co.). For the detection of NSM2 transcripts the following primers (Sigma-Aldrich) were used: Nsm2_fw: 5′-TGCTGGACACAAACGGTCT; Nsm2_rev: 5′ – GTTGTCCGGGGTACACACAT. The three housekeeping genes GAPDH, GUSB and HPRT1 were used as endogenous controls.
7
Genotyping SNPs in Epidermal Differentiation Complex
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Three SNPs were selected for genotyping that map to the 1q21 locus comprising Epidermal Differentiation Complex (EDC) (LCE3A: rs1886734, LCE3D: rs4112788; LCE3E: rs7516108). Two SNPs, rs1886734 and rs4112788, have previously shown to be significantly associated in Chinese population18 (link). Two independent studies reported rs4112788 to be the most associated variant18 (link)40 (link) and also to be in strong LD with LCE3C-LCE3B deletion40 (link). The third SNP (rs7516108), near the LCE3E gene was independent of LCE3C-3B deletion, showed significant association in the GWAS, however, failed to show any association in the follow up study among Chinese population18 (link). All SNPs were genotyped on a 7900HT Fast Real-Time PCR System Instrument using allele-specific Taqman MGB probes labelled with fluorescent dyes FAM and VIC (Applied Biosystems), following manufacturer’s protocols. Allelic discrimination was made with the ABI PRISM 7900HT SDS and the SDS 2.2.2 program (Applied Biosystems). Characteristics of the SNPs studied and their HWE status in our sample are presented in Table S3. Approximately 5% of the samples were randomly selected for Sanger sequencing to check for genotyping errors. More than 99% concordance was observed between these two methods.
8
Sensitive miRNA Detection and Quantification by qRT-PCR
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For the detection and quantification
of miRNA by qRT-PCR, we developed a novel protocol that uses commercially
available reagents. In order to fall within the dynamic range of the
standard curve, whole blood total RNA eluent was diluted 1:10–1:100000
for spike-in isolation recovery studies. The diluted RNA, RT primer
and water was heat-denatured at 90 °C for 1 min and then cooled
to 4 °C. After this initial heat-denaturing step, the remaining
RT reagents were added and the RNA was converted to cDNA using MMLV-RT
(Invitrogen) under the following conditions: 4 °C for 15 min,
16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5
min. TaqMan microRNA assays were used for expression analysis of hsa-miR-34a
and hsa-miR-24 (Applied Biosystems, Foster City, CA). Following cDNA
synthesis, qPCR was performed on 2 μL of cDNA on the ABI Prism
7900HT SDS (Applied Biosystems) using Platinum Taq Polymerase (Invitrogen)
under the following cycling conditions: 95 °C for 1 min (initial
denature) and then 50 cycles of 95 °C for 5 s and 60 °C
for 30 s. Additions to the manufacturers’ reagents include
DMSO (final concentration of 6%) and tetramethylammoniumchloride (TMAC;
final concentration of 50 mM in both RT and PCR) to improve the slope,
linearity, and sensitivity of the assay.
of miRNA by qRT-PCR, we developed a novel protocol that uses commercially
available reagents. In order to fall within the dynamic range of the
standard curve, whole blood total RNA eluent was diluted 1:10–1:100000
for spike-in isolation recovery studies. The diluted RNA, RT primer
and water was heat-denatured at 90 °C for 1 min and then cooled
to 4 °C. After this initial heat-denaturing step, the remaining
RT reagents were added and the RNA was converted to cDNA using MMLV-RT
(Invitrogen) under the following conditions: 4 °C for 15 min,
16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5
min. TaqMan microRNA assays were used for expression analysis of hsa-miR-34a
and hsa-miR-24 (Applied Biosystems, Foster City, CA). Following cDNA
synthesis, qPCR was performed on 2 μL of cDNA on the ABI Prism
7900HT SDS (Applied Biosystems) using Platinum Taq Polymerase (Invitrogen)
under the following cycling conditions: 95 °C for 1 min (initial
denature) and then 50 cycles of 95 °C for 5 s and 60 °C
for 30 s. Additions to the manufacturers’ reagents include
DMSO (final concentration of 6%) and tetramethylammoniumchloride (TMAC;
final concentration of 50 mM in both RT and PCR) to improve the slope,
linearity, and sensitivity of the assay.
9
Validating ZNF649 Variant Genotypes
10
Quantitative RT-PCR in Hippocampal Neurons
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Total RNA from hippocampal neurons or slices were extracted with Trizol Reagent (Ambion/RNA Life Technologies Co.) following the manufacturer procedures and cleaned up using RNeasy Mini kit (Qiagen, Hilden, Germany). RNA was quantified by absorbance at 260 nm using a Nanodrop ND-100 (Thermoscientific; Themo Fisher Scientific Inc.). Retrotranscription to first strand cDNA was performed using RevertAid H Minus First Strand cDNA Synthesis kit (Thermoscientific; Themo Fisher Scientific Inc.). Briefly, 5 ng of synthesized cDNA was used to perform fast qPCR using GoTaq qPCR Master Mix (Promega Co., Madison, WI, USA) in ABI PRISM 7900HT SDS (Applied Biosystems; Life Technologies Co.) with the manufacturer's protocol. The primers purchased to Sigma-Aldrich (Supplementary Table 13 ) were used at 0.5 μM final concentration to detect Bdnf transcripts and Jmjd3. Three housekeeping genes Gapdh, GusB and Pgk1 were used as endogenous controls.
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