Total RNA was isolated from the cells using RNAiso Plus (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. Total RNA (5 µg) was reverse transcribed into cDNA using the M-MLV First-Strand Synthesis system (Promega Corporation, Madison, WI, USA). cDNA was analyzed in triplicate using the MJ Real-Time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primers used are shown in Table I . qPCR was performed using the Power SYBR Green Master Mix (Takara Bio, Inc.) and the ABI 7300 Real-Time PCR detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR conditions were as follows: Initial denaturation step at 95°C for 15 sec, followed by 40 cycles of amplification and quantification at 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec. Fold changes in expression of each gene were calculated using the comparative quantification method (19 (link)).
Abi 7300 real time pcr detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Japan
The ABI 7300 real-time PCR detection system is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is capable of detecting and quantifying nucleic acid sequences in real-time during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Power sybr green master mix, by Takara Bio (4 mentions)
Rnaiso plus, by Takara Bio (3 mentions)
Amv reverse transcriptase, by Promega (3 mentions)
M mlv reverse transcriptase, by Promega (3 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Hiscript q rt supermix for qpcr kit, by Vazyme (2 mentions)
Sybr green master mix, by Vazyme (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Bulge loop mirna primer set, by RiboBio (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
High pure rna isolation kit, by Roche (2 mentions)
M mlv first strand synthesis system, by Promega (1 mentions)
Mj real time pcr system, by Bio-Rad (1 mentions)
Evagreen 2 qpcr mastermix, by Thermo Fisher Scientific (1 mentions)
All in one rt mastermix, by Thermo Fisher Scientific (1 mentions)
Amv reverse transcriptase, by Takara Bio (1 mentions)
28 protocols using abi 7300 real time pcr detection system
1
Quantifying Gene Expression via RT-qPCR
2
Quantification of Cytokine Expression
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The mammary gland tissues were homogenized in PBS, frozen and thawed three times; the supernatants were obtained after centrifugation at 9000× g for 20 min. The total RNA was then extracted from the supernatants of the mammary gland tissues by the TRIzol reagent, and the cDNA was generated by using a reverse transcription kit (Invitrogen, CA, USA) according to the manufacturer’s instructions. The specific primers of five cytokines were listed in Table 1 [35 (link),40 (link),41 (link),42 (link)]. Quantitative real-time PCR was performed on an ABI 7300 Real-Time PCR Detection System (Applied Biosystems, Foster, CA, USA) in a 20-μL reaction volume. Each sample was assessed in triplicate. The reaction volume and the reaction condition can be referenced from our lab’s previous studies [29 (link)]. The results (fold changes) were normalized by GAPDH using the 2−ΔΔCt comparative method [41 (link)].
3
Quantifying Stem Cell Markers by qRT-PCR
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Total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA (1 μg) was transcribed into cDNA with the 5× All-In-One RT MasterMix (Applied Biosystems, Foster City, CA, USA). The qRT-PCR was performed using an EvaGreen 2× qPCR MasterMix (Applied Biosystems, Foster City, CA, USA) and ABI 7300 real-time PCR detection system (Applied Biosystems, USA). The levels of mRNA expression for each gene were normalized by its respective GAPDH. Fold changes in gene expression were calculated by a comparative threshold cycle (Ct) method using the formula 2−(ΔΔCt). The PCR primers were synthesized by Beijing Genomics Institute (Beijing, China), and the primers used were as follows:
CD133-F, 5′-TACAACGCCAAACCACGACTGT-3′;
CD133-R, 5′-TCTGAACCAATGGAATTCAAGACCCTTT-3′;
CD44-F, 5′-GACACATATTGTTTCAATGCTTCAGC-3′;
CD44-R, 5′-GATGCCAAGATGATCAGCCATTCTGGAAT-3′;
ALDHA1-F, 5′-GCACGCCAGACTTACCTGTC-3′;
ALDHA1-R, 5′-CCTCCTCAGTTGCAGGATTAAAG-3′;
Oct4-F, 5′-TGGGATATACACAGGCCGATG-3′;
Oct4-R, 5′-TCCTCCACCCACTTCTGAG-3′;
Nanog-F, 5′-TTTGTGGGCCTGAAGAAAACT-3′;
Nanog-R, 5′-AGGGCTGTCCTGAATAAGCAG-3′;
β-catenin-F, 5′-AAGACATCACTGAGCCTCCAT-3′;
β-catenin-R, 5′-CGATTTGCGGGACAAAGGGCAA-3′;
PCNA-F, 5′-CTGAAGCCGAAACAGCTAGACT-3′;
PCNA-R, 5′-TCGTTGATGAGGTCTTGAGTGC-3′;
Cyclin D1-F, 5′-AGGCCCTGGCTGCTACAAG-3′;
Cyclin D1-R, 5′-ACATCTGAGTGGGTCTGGAG-3′;
GAPDH-F, 5′-CAAGGTCACCATGACAACTTTG-3′;
GAPDH-R, 5′-GTCCACCACCCTGTTGCTGTAG-3′.
4
Spatial and Temporal Transcriptome Profiling
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Total RNA samples were collected from the head, midgut, cuticula, fat body and trachea in 3L5 larvae for spatial transcript profile; every day from 1L1 larvae to 3L5 larvae for temporal transcript profile and from larvae subjected to 3-day’s bioassays for testing the relative expression of genes after RNAi-treatment. All samples were extracted by using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Each sample contained 5 larvae and replicated thrice. The house-keeping gene EF-1 was used as internal control58 (link). The primers of target genes were designed with Primer premier 5 (Table 3 ). The expression profiles of the four genes were determined using q-PCR with ABI 7300 Real-Time PCR detection system (Applied Biosystems Inc, Foster City, CA, USA). The PCR program included 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s and 72 °C for 31 s. The total reaction volume was 20 μl. All reactions were performed in triplicates.
5
Gene Expression Analysis of Epithelial-Mesenchymal Transition
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RNA extraction was carried out from T24 and EJ cells using RNAiso Plus (TaKaRa, Japan) in line with standard protocols. cDNA was synthesized from 2 mg of total RNA with AMV reverse transcriptase (Takara) in accordance with the manufacturer’s instructions. qRT-PCR was conducted using Power SYBR Green Master Mix (TaKaRa, Japan) with ABI 7300 real-time PCR detection system (Applied Biosystems, USA) under default conditions: 95°C for 10 s, and 40 cycles of 95°C for 10 s and 72°C for 30 s. Comparative Ct method was employed for quantification, and the mRNA expression of every gene was normalized to its respective GAPDH. The primers used were as follows: GAPDH, forward 5′-GCTGCCCAACGC ACCGAATA-3′ and reverse 5′-GAGTCAACGGATTTGGTCGT-3′; ZO-1, forward 5′-GCAGCCACAACCAATTCATAG-3′ and reverse 5′-GCAGACGATGTTCATAGTTTC-3′; E-cadherin, forward 5′-TCGACACCCGATTCAAAGTGG-3′ and reverse 5′-TTCCAGAAACGGAGGCCTGAT-3′; N-cadherin forward 5′-ATCAAGTGCCATTAGCCAAG-3′ and reverse 5′-CTGAGCAG TGAATGTTGTCA-3′; Vimentin, forward 5′-CCTTGACATTGA GATTGCCA-3′ and reverse 5′-GTATCAACCAGAGGGAGTGA-3′; Snail, forward 5′-TTCCAGCAGCCCTACGACCAG-3′ and reverse 5′-CGGACTCTTGGTGCTTGTGGA-3′.
6
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated by RNAiso Plus according to the manufacturer's instructions (TaKaRa, Japan). qRT-PCR was performed using Power SYBR Green Master Mix (TaKaRa, Japan) and an ABI 7300 real-time PCR detection system (Applied Biosystems). Fold changes in expression of each gene were calculated by a comparative threshold cycle (Ct) method using the formula 2− (ΔΔCt).
7
Quantifying miRNA Expression in Differentiating Cells
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To analyze miRNA gene expression levels, quantitative real time PCR was performed on differentiating cultures at day 6 and day 10. 1 µg of RNA was reversed transcribed using SuperScript® III (Invitrogen) and miRNA expression levels were determined using Taqman Gene Expression Assays with Taqman Universal PCR Master Mix (ThermoFisher) on the ABI 7300 Real-Time PCR detection system (Applied Biosystems). Transcript expression levels were normalized to the averaged expression of the reference gene snoRNA RNU24, and gene relative quantification was calculated using the 2−∆∆Ct method.
8
Validated Differential Protein Expression
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To validate the differential proteins, quantitative real-time PCR (qPCR) was used
to confirm the expression patterns of selected proteins in
Bnclv-like and Ningyou12. The total RNA of collected
samples were extracted using TRIzol reagent (Life technologies, USA) following
the protocol of the supplier. First strand cDNA was synthesized by reverse
transcription of total RNA (500 ng) using the HiScript Q RT SuperMix for qPCR
kit (Vazyme, China). All reactions were performed with an ABI 7300 Real-Time PCR
Detection System (Applied Biosystems, USA) with SYBR Green Master Mix (Vazyme,
China). Primer premier 5.0 was used to design gene-specific primers according to
the corresponding unigene sequences. The sequences of primers were listed in
Table
S1 . Primers were checked for efficiency
using the standard curve method, and their specificities were checked using
melting curves after all qPCR runs. All qPCRs were performed in triplicate in a
total volume of 20 μL. The ACTIN gene was used as an internal
reference gene. The relative expression levels of genes were calculated using
the 2-ᐃᐃCt method.
to confirm the expression patterns of selected proteins in
Bnclv-like and Ningyou12. The total RNA of collected
samples were extracted using TRIzol reagent (Life technologies, USA) following
the protocol of the supplier. First strand cDNA was synthesized by reverse
transcription of total RNA (500 ng) using the HiScript Q RT SuperMix for qPCR
kit (Vazyme, China). All reactions were performed with an ABI 7300 Real-Time PCR
Detection System (Applied Biosystems, USA) with SYBR Green Master Mix (Vazyme,
China). Primer premier 5.0 was used to design gene-specific primers according to
the corresponding unigene sequences. The sequences of primers were listed in
S1
using the standard curve method, and their specificities were checked using
melting curves after all qPCR runs. All qPCRs were performed in triplicate in a
total volume of 20 μL. The ACTIN gene was used as an internal
reference gene. The relative expression levels of genes were calculated using
the 2-ᐃᐃCt method.
9
Quantification of miR-497 Expression
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Total RNA was extracted from cells with isolator reagent (Vazyme, China). After measurement of the RNA concentration, cDNAs were generated from reverse transcription with the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). The expression level of miR-497 was measured according to the instructions of the ChamQTM Universal SYBR qPCR Master Mix kit (Vazyme, China) using the ABI-7300 Real-Time PCR Detection System (Applied Biosystems, USA). The bulge-loopTM miRNA Primer Sets (one RT primer and a pair of qPCR primers) specific for miR-497 were purchased from RiboBio (Guangzhou, China). The level of miR-497 was normalized to U6. Fold changes were calculated using the 2−ΔΔCt method. Each plate was run in triplicate.
10
RNA Extraction and Real-Time PCR Analysis
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RNA extraction was conducted using TRIzol reagent (Invitrogen). Real-time PCR was performed as previously reported5 using an ABI 7300 real-time PCR detection system (Applied Biosystems, Foster City, CA, USA) and Power SYBR Green Master Mix (TaKaRa, Kusatsu, Shiga, Japan). Reverse transcription of RNA into cDNA was performed using reverse transcriptase (Promega, Madison, WI, USA). GAPDH expression served as the loading control in real-time PCR. The primers used for PCR are presented in Table 1 .
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