Tissues (∼100 mg) were homogenized in liquid nitrogen with a glass homogenizer. Total RNA was extracted using QIAzol® Lysis Reagent (QIAGEN Science, Germantown, MD, USA) according to the manufacturer's instructions. RNA was converted into complementary DNA (cDNA) using a RevertAid™ First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Next, qRT-PCR was conducted using an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). Twenty microliters of reaction volume contained 10 μl SYBR Green Master Mix (New England Biolabs, Ipswich, MA, USA), 4 μl cDNA and 200 nmol/l primer set. The PCR reactions were conducted as follows: 2 min at 50°C, 10 min at 95°C and 40 cycles at 95°C for 15 s, followed by 1 min at 60°C. The relative expression levels were determined as the Δcycle threshold (ΔCt). All primer sets used in the present study are shown in Table S1 .
Abi prism 7500 sequence detection
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 7500 is a real-time PCR system designed for gene expression analysis, SNP genotyping, pathogen detection, and other applications. It uses fluorescent dyes to detect and quantify DNA sequences during the amplification process. The system includes a thermal cycler, an optical detection module, and data analysis software.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by New England Biolabs (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Nuclear and cytoplasmic extraction reagents kit, by BestBio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
Quantifast probe pcr kit, by Qiagen (1 mentions)
Prim scripttm rt reagent kit, by Takara Bio (1 mentions)
Allprep dna rna protein mini kit, by Qiagen (1 mentions)
Human β actin, by Thermo Fisher Scientific (1 mentions)
Power sybr green master mix reagent, by Thermo Fisher Scientific (1 mentions)
High capacity cdna transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Power sybr green pcr master mix, by Roche (1 mentions)
Reverse transcription system, by Roche (1 mentions)
Kapa sybr fast universal, by Roche (1 mentions)
Sybr green pcr mix, by Roche (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
8 protocols using abi prism 7500 sequence detection
1
RNA Extraction and qRT-PCR Analysis
2
Transcriptome Analysis by qRT-PCR
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Total RNA isolation was done with the TRIzol reagent (Invitrogen, USA). After quality control and quantification, individual RNA samples (1μg each) were converted to cDNA with the Prime Script RT reagent Kit (Takara, Japan), and detected by Hieff® qPCR SYBR Green Master Mix (Yeasen, China) on ABI Prism 7500 sequence detection system (Applied Biosystems, USA). The nuclear and cytoplasmic fractions were isolated with a BestBio Nuclear and Cytoplasmic Extraction Reagents Kit (BestBio, China). GAPDH and small nuclear U6 were employed as endogenous controls. qRT‐PCR was conducted using primers obtained from Tsingke (Changsha, China). The primer sequences used are described in Table S1 .
3
Quantifying EphA5 mRNA Expression
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Total RNA was extracted from cells and tissues using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Germany), and samples were reverse transcribed using the PrimScriptTM RT Reagent Kit (TaKaRa, China) as described in the manufacturer’s protocol. Real-time PCR to determine EphA5 and GAPDH mRNA levels was performed using the QuantiFast Probe PCR Kit (Qiagen, Germany) according to the manufacturer’s instructions; all analyses were conducted using the ABI Prism 7500 sequence detection system (Applied Biosystems, CA). The relative quantification of EphA5 mRNA levels was performed using the comparative Ct method (2-△△Ct method) with GAPDH as the reference gene. Increases or decreases in mRNA levels of at least two fold were considered to be significant. The primer sequences were as follows: EphA5 forward, 5’-TCTGTGGTACGACACTTGGC-3’; EphA5 reverse, 5’-CTTGCACATGCATTTCCCGA-3’; GAPDH forward, 5’-GAGAAGGCTGGGGCTCATTT-3’; GAPDH reverse, 5’-AGTGATGGCATGGACTGTGG-3’.
4
Quantitative RT-PCR Analysis of Survivin
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We isolated total RNAs from tumor tissues, cells or implanted tumors using an RNeasy Mini kit, according to the manufacturer's protocol (Qiagen, Valencia, CA, USA), and first-strand cDNAs were synthesized using a High Capacity cDNA Transcription kit (Applied Biosystems, Foster City, CA, USA). qPCR was performed in a 20-μl reaction mixture using the Power SYBR Green Master Mix reagent (Applied Biosystems) on an ABI PRISM 7500 sequence detection system (Applied Biosystems). The cycling conditions were as follows: 1 cycle at 95°C for 10 min followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Primers for human survivin [5′-CTT GGC CCA GTG TTT CTT CT-3′ (upstream) and 5′-CCT CCC AAA GTG CTG GTA TT-3′ (downstream)] and the internal control, human β-actin [5′-AGT CCT GTG GCA TCC ACG AAA-3′ (upstream) and 5′-GTC ATA CTC CTG CTT GCT GA-3′ (downstream)] were synthesized by Applied Biosystems. The values were normalized with β-actin, and relative expression was analyzed using the ΔΔCt method.
5
RNA Isolation and Gene Expression Analysis
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Total cellular RNA was isolated with TRIzol reagent (Invitrogen) and used for first-strand cDNA synthesis with the Reverse Transcription System (Roche). Quantitation of all gene transcripts was performed by qPCR using a Power SYBR Green PCR Master Mix (Roche) and an ABI PRISM 7500 sequence detection system (Applied Biosystems) with the expression of GAPDH as the internal control. The qRT-PCR primers are shown in Supplementary Table S5 .
6
Quantifying lncRNA Expression in Whole Blood
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By using TRIzol reagent (Invitrogen), total cellular RNA was isolated from peripheral whole blood samples. cDNA was synthesized with the Takara PrimeScript™ RT Master Mix Kit (Shiga, Japan). Using KAPA SYBR Fast Universal (Kapa Biosystems, Wilmington, MA, USA), we performed qPCR to evaluate lncRNA expression. The ABI Prism 7500 sequence detection system was used to perform qPCR (Applied Biosystems, Foster City, CA, USA). Briefly, the reactions were performed in a mixture (20 μL) containing 8 μL H2O, 10 μL 2X SYBR-Green PCRMix (Kapa), 1 μL cDNA template, and 0.5 μL each of sense and antisense primers. A total of 3 lncRNAs were tested. Primer sequences are listed in Table 2 . We used GAPDH as a housekeeping gene for normalization. The relative changes in lncRNA expression were calculated using the 2−ΔΔCt method. When the Eos asthma patient was cured, another 2 mL of blood was collected for verification of specific genes by RT-PCR. Statistical analysis was conducted as described above.
7
Quantifying FKBP9P1 Expression in Tumor Samples
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Total RNA was isolated from tissues or cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reversed transcribed into cDNAs using Transcriptor First Strand cDNA synthesis kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. The mRNA level was measured using the SYBR Green fluorescence kit (Roche) and the ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). The expression of FKBP9P1 was normalized to housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The ratio of relative mRNA expression between the tumor (experimental) group and the normal (control) group was calculated based on the 2−ΔΔCt method. The formula is as follows: ΔΔCt = ΔCtexperimental group − △Ctcontrol group, △Ct = Cttarget gene − CtGAPDH. Ct represents the amplification cycles when real-time fluorescence intensity reached threshold at reaction. The experiment was performed in triplicate. The following primers were used for qRT-PCR. FKBP9P1 forward: 5′-TCTTCTCATAGGAACACTCTCAGT-3′; FKBP9P1 reverse: 5′-CTCGCCAACGCACATCTC-3′; GAPDH forward: 5′-GATCATCAGCAATGCCTCCT-3′; GAPDH reverse: 3′-TGAGTCCTTCCACGATACCA-5′.
8
Quantitative Expression Analysis of S100A4
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Total RNA was extracted by TRIzol, and cDNAs were reverse transcribed by RevertAid TM reverse transcriptase (Takara). Real-time PCR was carried out using the ABI PRISM 7500 Sequence detection system (Applied Biosystems). The primers for GAPDH (135bp, 201-335, NM_ 002046.3) were 5'-CAATGACCCCTTCATTGACC-3' (sense) and 5'-TGGAAGATGGTGATGGGATT-3' (antisense). The primers for S100A4 (212bp, 333-525, NM_005978.3) were 5'-TCCAAGAGTAC TGTGTCTTC-3' (sense) and 5'-TATTGAACTTGCTCAGCATC-3' (antisense). Expression of GAPDH was used to normalize that of the target genes. Each assay was done in triplicate, the average was calculated, and the expression level of S100A4 was expressed as 2 -∆Ct , where ∆Ct=Ct (S100A4)-Ct (GAPDH).
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