Total RNA was extracted from 4‐μm‐thick FFPE specimens by manual microdissection using RNeasy FFPE Kit (Qiagen). The complementary DNA (cDNA) synthesis was performed using PrimeScript RT Master Mix (RR036A) (TAKARA). The quantitative reverse‐transcriptase polymerase chain reaction (RT‐PCR) assays were performed using ViiA 7 Dx RT‐PCR System (Applied Biosystems) using PowerUp SYBR Green Master Mix (Applied Biosystems). The cycling conditions were as followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The relative expression of target genes was normalized against glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) using the 2−ΔCT method.
Viia 7 dx rt pcr system
Manufactured by Thermo Fisher Scientific
The ViiA 7 Dx RT-PCR System is a real-time PCR instrument designed for diagnostic applications. It features a compact design and supports multiple sample formats. The system enables rapid and sensitive detection and quantification of nucleic acid targets.
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Lab products found in correlation
4 protocols using viia 7 dx rt pcr system
1
FFPE RNA Extraction and RT-qPCR Analysis
2
RNA Extraction and qRT-PCR Analysis of FFPE Specimens
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We extracted total RNA via the RNeasy FFPE Kit (Qiagen, #73504) from 4‐μm‐thick FFPE specimens. We used PrimeScript RT Master Mix (Takara, #RR035A) reverse transcription of the complementary DNA (cDNA). We performed the qRT‐PCR assays using the ViiA 7 Dx RT‐PCR System (Applied Biosystems) with AceQ qPCR SYBR Green Master Mix (Vazyme, #Q111‐02). The internal control of target genes GAPDH using the 2^‐ΔΔCT method. The primer sequences are provided in Table S2 . We used ‘combat’ function from ‘sva’ R package (version 3.36) to remove the batch effects.
3
Quantifying gene expression changes
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Cells grown in 6-well plates from the two groups were washed and RNA was extracted using RNAzol (GeneCopoeia, Inc.). According to the manufacturer's protocol, an All-In-One™ First-Strand cDNA Synthesis kit (cat. no. AORT-0020; GeneCopoeia, Inc.) was used for RT. Briefly, 1 µg total RNA was used for DNA synthesis. qPCR was performed as follows: Initial denaturation at 95°C for 10 min; followed by 40 cycles at 95°C for 10 sec, 60°C for 20 sec and 72°C for 34 sec using an ViiA 7 Dx RT-PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The mRNA expression levels of GAPDH were used as an internal control. Changes in expression were determined using the 2−ΔΔCq method (17 (link)). The primer sequences used included: Nefh (2 µM; cat. no. RQP048942; GeneCopoeia, Inc.) and GAPDH (2 µM; cat. no. HQP006940; GeneCopoeia, Inc.).
4
STAD Surgical Protocol with qRT-PCR
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Twelve patients who were subject to surgery without neoadjuvant chemotherapy (NAC) and were diagnosed with STAD at Affiliated Haian Hospital of Nantong University. The study was permitted by the Regional Ethics Committee at Affiliated Haian Hospital of Nantong University. The experiments were conducted with the understanding and written consent of each participant. The study methodologies conformed to the standards set by the Declaration of Helsinki. Study material consisted of formalin‐fixed paraffin‐embedded (FFPE) specimens obtained from radical surgery from 2017 to 2020. Each patient were subjected to a standard radical surgical procedure, and all specimens were analyzed by expert pathologists. Total RNA was extracted from FFPE slides using RNeasy FFPE Kit (Qiagen). The synthesis of complementary DNA (cDNA) was carried out by PrimeScript RT Master Mix (RR036A) (TAKARA). We conducted the quantitative reverse‐transcriptase polymerase chain reaction (qRT‐PCR) assays by ViiA 7 Dx RT‐PCR System (Applied Biosystems) with PowerUp SYBR Green Master Mix (Applied Biosystems). The cycling conditions and procedure were followed as 40 cycles of 95°C for 15 s and 60°C for 60 s. The relative expression of target genes (2−ΔCT) was normalized against GAPDH reference gene.
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