Plasma viral RNA (vRNA) levels were determined by qRT-PCR (ABI Prism 7900 sequence detection system; Applied Biosystems). vRNA was reverse transcribed and cDNA amplified (45 cycles/default setting) with AmpliTaq Gold DNA polymerase (PCR core reagents kit; Perkin-Elmer/Roche) utilizing primer pairs corresponding to SIVmac239 gag gene sequences (forward, nucleotides 1181–1208, and reverse, 1338-1317). Cell-associated viral DNA within sorted CD4 TM cells was measured as previously20 (link). Briefly, qRT-PCR was performed on sorted, lysed cells using the Taq DNA polymerase kit (Invitrogen) and the SIVmac239 forward primer GTCTGCGTCATYTGGTGCATTC, reverse primer CACTAGYTGTCTCTGCACTATRTGTTTTG, and probe sequence CTTCRTCAGTYTGTTTCACTTTCTCTTCTGCG.
Abi prism 7900 sequence detection
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 7900 sequence detection system is a real-time PCR instrument designed for high-throughput nucleic acid quantification and detection. The core function of this product is to provide accurate and reliable real-time PCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
Taq dna polymerase kit, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Miscript reverse transcription kit, by Qiagen (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
5 protocols using abi prism 7900 sequence detection
1
Quantification of Viral RNA and DNA in SIV-Infected Samples
2
Quantification of Viral RNA and DNA in SIV-Infected Samples
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Plasma viral RNA (vRNA) levels were determined by qRT-PCR (ABI Prism 7900 sequence detection system; Applied Biosystems). vRNA was reverse transcribed and cDNA amplified (45 cycles/default setting) with AmpliTaq Gold DNA polymerase (PCR core reagents kit; Perkin-Elmer/Roche) utilizing primer pairs corresponding to SIVmac239 gag gene sequences (forward, nucleotides 1181–1208, and reverse, 1338-1317). Cell-associated viral DNA within sorted CD4 TM cells was measured as previously20 (link). Briefly, qRT-PCR was performed on sorted, lysed cells using the Taq DNA polymerase kit (Invitrogen) and the SIVmac239 forward primer GTCTGCGTCATYTGGTGCATTC, reverse primer CACTAGYTGTCTCTGCACTATRTGTTTTG, and probe sequence CTTCRTCAGTYTGTTTCACTTTCTCTTCTGCG.
3
Total RNA Isolation and qPCR Analysis
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The TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was employed for the total RNA isolation from tissue specimens and cells. To detect LINC00958 and NUAK1 mRNA expression, reverse transcription was conducted to convert total RNA to cDNA with the PrimeScript RT-Reagent Kit (Takara Bio, Kusatsu, Japan). Subsequently, the amplification reaction was carried out using the SYBR Premix Ex Taq™ Kit (Takara Bio) on an ABI Prism 7900 sequence detection system (Applied Biosystems Inc.). LINC00958 and NUAK1 mRNA levels were normalized to GAPDH. To quantify miR-625 expression, the synthesis of cDNA was performed with the miScript Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany), and the cDNA was then subjected to qPCR by means of the miScript SYBR Green PCR Kit (Qiagen GmbH). U6 small nuclear RNA served as an internal control of miR-625 expression. The 2−ΔΔCq method was employed to analyze relative gene expression.
4
Quantitative Analysis of MMP-9 Expression
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Total RNA was isolated using TRIzol® Reagent (Life Technologies, Grand Island, USA). The cDNA was synthesized from 1 µg total RNA by using the PrimeScript™ RT reagent kit (TaKaRa, Shiga, Japan). The RNA expression levels of MMP-9 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were determined using the ABI PRISM 7900 sequence detection system and SYBR® Green (Applied Biosystems, Foster City, USA). The following primers (2 µM) were used: MMP-9 (NM 004994), sense 5´-CCTGGAGACCTGAGAACCAATCT-3´, antisense 5´-CCACCCGAGTGTAACCATAGC-3´; and GAPDH (NM 002046), sense 5´-ATGGAAATCCCATCACCATCTT-3´, antisense 5´-CGCCCCACTGATTTTGG-3´.
5
mRNA Expression Analysis of Schizophrenia
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The mRNA expression analysis included 45 Japanese patients with schizophrenia and 45 age- and sex-matched healthy Japanese controls (Supplementary Table 1 ). Total RNA was extracted from immortalized lymphocytes from the schizophrenia patients and the healthy controls using an RNeasy Mini kit (QIAGEN K.K., Tokyo, Japan). ARHGAP33 and SORT1 mRNA expression levels were measured via real-time quantitative RT–PCR using an ABI Prism 7900 sequence detection system (Applied Biosystems), as described previously54 (link). The TaqMan Pre-Developed Assay Reagent kit (Applied Biosystems) was used for the mRNA expression analysis of GAPDH (4326317E), ARHGAP33 (Hs00364775_m1) and SORT1 (Hs00361747_m1). PCR data were obtained using Sequence Detector Software (SDS version 2.1, Applied Biosystems) and were quantified according to a standard curve method. The expression levels of ARHGAP33 and SORT1 were normalized to GAPDH mRNA.
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