Total RNA from plasma and the hippocampus were extracted by TRIZOL® reagent (Invitrogen) according to the manufactory manual. The RNA samples were dissolved in 50 μL of DEPC‐H2O and subsequently stored at −80°C. The quantification and purity of the RNA were measured by NanoDrop2000 (Thermo‐Fisher Scientific). Total RNA was transcribed by a HiFiScript cDNA Synthesis Kit (CWBIO) according to the manufactory protocol. Real‐time PCR was performed on the ABI7300 platform (Applied Biosystems, Life Technologies) by using ChamQ SYBR qPCR Master Mix (Vazyme). Data were analyzed using the 2‐ΔΔCt method. Primers for each RNA were listed in Table S1 .
Abi7300 platform
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI7300 platform is a real-time PCR system designed for gene expression analysis. It features a 96-well block format and supports a range of fluorescent chemistries, including TaqMan, SYBR Green, and Molecular Beacons. The system is capable of performing relative and absolute quantification of gene expression targets.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Hifiscript cdna synthesis kit, by CWBIO (1 mentions)
Chamq sybr qpcr master mix, by Vazyme (1 mentions)
Trizol, by Takara Bio (1 mentions)
First strand synthesis kit, by Roche (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Sybr green master mix, by Toyobo (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Mgb taqman probes, by Thermo Fisher Scientific (1 mentions)
Taqman genotyping master mix, by Thermo Fisher Scientific (1 mentions)
Uv 1700 pharmaspec, by Shimadzu (1 mentions)
8 protocols using abi7300 platform
1
Plasma and Hippocampus RNA Extraction and Quantification
2
Quantitative Analysis of TMEM106C Expression
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TRIzol (Takara, Dalian, China) was used to extract total RNA from tissues. Total RNA was reverse transcribed to generate cDNA. TMEM106C mRNA levels were examined using a SYBR Green-based real-time PCR kit (Vazyme Biotech, Nanjing, China) on an ABI 7300 Platform (Applied Biosystems, CA). Data were analyzed based on the comparative cycle threshold (Ct) values. The primer sequences of TMEM106C, GAPDH, CENPM, and DLC-1 can be found in Supplementary Table 2 .
3
Quantitative Expression Analysis of RNAs
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Total RNA was isolated from clinical specimens or cell lines using the TRIzol reagent (TaKaRa Biotechnology Co., Ltd., Cat#9109, Dalian, Liaoning, China). cDNA was synthesized using a First-Strand Synthesis Kit (Roche Diagnostics, Cat# 04896866001, Indianapolis, IN, USA). qRT-PCR was performed using a FastStart Universal SYBR Green Master (ROX, Roche Diagnostics, Cat# 04707516001) on an ABI7300 platform (Applied Biosystems, Foster City, CA, USA). The expression of mRNAs and MALAT1 was normalized to GAPDH, while miRNA was normalized to U6. The sequences of specific primers are listed in Supplementary Table 5 , part of which was designed according to previous reports [43 (link)]. The data were calculated with the 2-ΔΔCt method [44 (link)].
4
Quantitative PCR for M. leprae
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DNA was extracted from dermal smear samples and biopsies (nerve and overlying skin) and detected by quantitative real-time polymerase chain reaction (qPCR) primer/probe assay targeting the M. leprae species-specific genomic region (RLEP3) (14 (link)). The reactions were performed on the ABI7300 platform (Applied Biosystems) and the result was analyzed using the 7,300 System SDS Software vs. 1.4.
5
Comprehensive Liver Disease Profiling
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Demographic and laboratory data were collected prior to liver biopsy. We recorded age, sex, white blood cell (WBC) count, platelet (PLT) count, prothrombin time (PT), the international standardization ratio (INT), and levels of albumin (ALB), globulin (GLB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), and serum total bilirubin (TBIL). Hepatitis B surface antigen (HBsAg), hepatitis B surface E antigen (HBeAg), and core antibody (anti-HBC) were detected using the CLIA system. The serum load of HBV-DNA was assessed via real-time polymerase chain reaction (ABI 7300 platform, Applied Biosystems, Foster City, CA, USA).
6
Quantifying Gene Expression in Inflorescences
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Total RNA extraction was performed using TRIzol (Life Technologies) following the protocol in the user’s manual. cDNAs of wild-type and pat inflorescences were used for the expression analysis of selected genes. Real-time quantitative PCR was performed using gene-specific primers and SYBR Green Master Mix (TOYOBO) on the ABI 7300 platform (Applied Biosystems). The experiments were repeated three times, and the data were averaged. The β-tubulin gene was used as an internal normalization control [65 (link)]. Fold changes in gene expression were calculated using the ΔΔCt (cycle threshold) values. The relevant primers are listed in S1 Table .
7
Comprehensive Liver Disease Assessment
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Clinical and laboratory parameters of patients were retrospectively collected from electronic medical records, including age, gender, white blood cell (WBC), platelet count (PLT), prothrombin time (PT), international normalized ratio (INR), albumin (ALB), globulin (GLB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), glutamyl transpeptidase (GGT), serum total bilirubin (TBIL), HBsAg, and hepatitis B surface E antigen (HBeAg) status. Core antibody (anti-HBC) was detected using the CLIA system. The serum load of HBV-DNA was assessed via real-time polymerase chain reaction (ABI 7300 platform, Applied Biosystems, Foster City, CA, USA). Blood tests of the cohort were obtained on the day before liver biopsy.
8
Caffeine Synthase Genotyping Protocol
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Genomic DNA was extracted from collected leaves using a CTAB based protocol (Doyle and Doyle 1990) . After extraction, DNA was quantified by absorbance under UV light at 260 and 280 nm on a spectrophotometer PharmaSpec UV-1700 (UV-Visible Spectrophotometer SHIMADZU), according to Sambrook et al. (1989) . A conventional PCR was performed to verify overall DNA quality using control actin specific primers as previously described (Maluf et al. 2009) .
Genotyping was performed by quantitative PCR using a combination of primers and specific MGB TaqMan probes (Life Technologies). Caffeine synthase specific primers and fluorescent probes annealing to a region with two SNPs were used for allelic discrimination using an ABI7300 platform (Applied Biosystems). Sequences and fluorescence used are listed in Online resource 2. For each reaction 100 ng of DNA were used, and amplification conditions were as suggested on the commercial TaqMan® Genotyping Master Mix (Thermo Scientific) and equipment manuals. All reactions were performed in duplicate, and were repeated two times for each genotype. Post-running analyses were performed using the software 7300 System SDS (Applied Biosystems).
Genotyping was performed by quantitative PCR using a combination of primers and specific MGB TaqMan probes (Life Technologies). Caffeine synthase specific primers and fluorescent probes annealing to a region with two SNPs were used for allelic discrimination using an ABI7300 platform (Applied Biosystems). Sequences and fluorescence used are listed in Online resource 2. For each reaction 100 ng of DNA were used, and amplification conditions were as suggested on the commercial TaqMan® Genotyping Master Mix (Thermo Scientific) and equipment manuals. All reactions were performed in duplicate, and were repeated two times for each genotype. Post-running analyses were performed using the software 7300 System SDS (Applied Biosystems).
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