CD71+ cells from murine bone marrow were sorted (100 cells/well) in 96-well plates containing 4ml/well of lysis buffer, and were snap frozen to −80°C. Control wells with 0, 20, 50 or 100 cells were included. Thawed plates were processed according to published protocol [23 (link)] where cDNA was pre-amplified in multiplex using gene specific Taqmanprimers and Taq/SSIII reaction mix (Invitrogen, Waltham, US), with an initial 50°C incubation step for the reverse transcription (RT). Gene expression analysis simultaneously on diluted samples was enabled using the 48.48 Dynamic array (Fluidigm, South San Francisco, US) and the Biomark HD platform (Fluidigm). The RT quantitative PCR (RT-qPCR) was run according to published protocol. RNA extraction was done using the RNeasy micro kit (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. Analysis was performed on a 7900 HT fast RT-PCR (ThermoFisher Scientific) and the primers used were Actin (#Mm02619580_g1), and HRI (#Mm01202300_m1), also from Thermi Fischer Scientific.
7900 ht fast rt pcr
Manufactured by Thermo Fisher Scientific
Sourced in Singapore
The 7900 HT Fast RT-PCR is a real-time PCR system designed for high-throughput gene expression analysis. It features fast thermal cycling capabilities and supports a wide range of sample volumes and plate formats. The system is capable of performing reverse transcription and PCR amplification in a single instrument.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Taqman primer, by Thermo Fisher Scientific (1 mentions)
Taq ssiii reaction mix, by Thermo Fisher Scientific (1 mentions)
Biomark hd platform, by Standard BioTools (1 mentions)
48.48 dynamic array, by Standard BioTools (1 mentions)
Sybr green mix, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Revertaid reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Primescript rt pcr kit, by Takara Bio (1 mentions)
Taqman, by Thermo Fisher Scientific (1 mentions)
6 protocols using 7900 ht fast rt pcr
1
Quantitative Gene Expression in Murine Cells
2
Quantification of CASC2 and miR-155 in PTC
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For detection of the abundances of CASC2 and miR-155 in PTC tissues and cells, Trizol reagent (Thermo Fisher) was used for total RNA isolation. Then, 1 μg RNA was reversely transcribed to cDNA using TaqMan microRNA reverse transcription kit or High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher).The cDNA was mixed with SYBR Green Mix (Thermo Fisher) and specific primers and used for qRT-PCR on a 7900HT Fast RT-PCR. The amplification procedure was as follows: 95°C for 5 min and 40 cycles at 95°C for 10 s, 60°C for 30 s. The primers were listed as follows: CASC2 (forward, 5′-GGCTCACAAAGCCTAGGTTA-3′; reverse, 5′-CCTTGGATATTTCCAAGAGC-3′); miR-155 (forward, 5′-CCCCACAGTCTACTGTAAG-3′; reverse, 5′-GCATTGCCGATGGTACTGATT-3′). GAPDH (forward, 5′-AGAAGGCTGGGGCTCATTTG-3′; reverse, 5′-AGGGGCCATCCACAGTCTTC-3′) and U6 (forward, 5′-CTCGCTTCGGCAGCACA-3′; reverse, 5′-AACGCTTCACGAATTTGCGT-3′) were used as internal controls. The relative levelsof CASC2 and miR-155 were calculated by the 2-ΔΔCt method [17 (link)].
3
Alkaline Stress Response in Rice
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Seeds of rice genotype (IRRI6) were sterilized with 2.5% sodium hypochlorite for 35 min followed by three washes and placed in 37°C Owen for 1 day. Uniformly germinated seeds were grown under normal conditions (16-h light/8-h dark at 26 ± 2°C) for 2 weeks. For this purpose, 96-wells plates supported by a container filled with nutrients media (Yoshida) were used. After this, one batch was kept as a control and in other batch 0.5% Na2CO3 solution (pH = 11.3) was applied for 36 h to create alkaline stress. The leaf samples were collected in triplicates. The total RNA was extracted with the help of TRIzol method and complementary DNA was prepared by using reverse transcriptase-III, first strand cDNA Synthesis Kit (K1691, Thermo Scientific Revert Aid). Real-time PCR for quantification (qRT-PCR) of selected genes of PPR, OZ1, and MORF/RIP gene family members was performed by using StepOne RT-PCR (Applied Biosystems® 7900 HT Fast RT-PCR). Three biological replicates from control and treated samples were used. OsActin was used as an internal control and for expression calculations 2ΔΔ CT method was used (Uzair et al., 2021 (link)). List of gene-specific primers are provided in Supplementary Table 1 .
4
Root Gene Expression Analysis
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Plants were harvested for RNA sampling after 15 days of treatment. Two biological replicates from each treatment of both cultivars were retained for RNA sampling. Primary roots of each plant were cut with sterile scissors and cleaned with 70% ethanol. Samples were enveloped in aluminum foil and immediately placed in liquid nitrogen. RNA extraction from the root samples (2 g) was performed using the TRIzol® method according to [36 ] with the Na-precipitation method. Both biological replicates were pooled after RNA extraction. After normalization, complementary DNA synthesis was performed using a Thermo Scientific Revert Aid-Reverse Transcriptase kit (K1691), according to the manufacturer’s protocol. Further, real-time quantitative PCR (Applied Biosystems® 7900 HT Fast RT-PCR) with StepOnePlus software was used to check the expression of the selected orthologs among three P treatment growth conditions (C, −P, and +P). SYBER-Green kit (Taq Man) was used for making reaction mixture. Both Actin and Tubulin reference genes were initially used, and both demonstrated a similar expression pattern. Hence, for further RT-PCR experiment reference gene TaActin was used to normalize the relative expression data. Relative expression values were evaluated by using the 2ΔΔCT method [37 (link)].
5
Extraction and Analysis of circRNA
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Total RNA was extracted using TRIzol reagent (Invitrogen, USA). Nuclear and cytoplasm RNA was extracted from MCF-10A and MDA-MB-231 cells using Paris™ kit (Thermo Fisher Scienti c, Massachusetts, USA). cDNA was generated by reverse transcription using the PrimeScript RT-PCR kit in accordance with the manufacturer's instructions (Takara, Tokyo, Japan). Real-time PCR was performed on a 7900HT Fast RT-PCR instrument (Applied Biosystems, Singapore). The primer of circ-LATS2 and miR-4686 were synthetized by Intergrated Biotech Solutions (Shanghai, China). The qRT-PCR results were analysed with the 2 -△△CT method.
6
Real-time qPCR for c-kit+ Fetal Liver Cells
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For real-time (RT) qPCR, 60,000 c-kit+ fetal liver cells were seeded in each well of a 96-well plate in the presence or absence of doxycycline. Harmine, dissolved in DMSO, was added at a concentration of 10 µM. Control wells received only DMSO. Twenty-four hours after adding Harmine, cells were frozen with 300 µL RLT buffer. RNA extraction was done using RNeasy micro kit (Qiagen, Venlo, the Netherlands) according to manufacturer's instructions. One hundred microliter reactions were set up using SYBR Green PCR buffer, in which the following primers were used: actin, F:5′-ATGGTGGGAATGGGT CAGAA-3′, R:5′CCATGTCGTCCCAGTTGGTAA-3′ and Rps19, F:5′-GCAGAGGCTCTAAGAGTGTGG-3′, F:5′-GCAGAGGCTCTAAGAGTGTGG-3′, R:5′-CCAGGTCT CTCTGTCCCTGA-3′.
When using the Taqman (Life Technologies, Carlsbad, CA), the following primers were used: p21 (00355782_m1) and Bax (00180269_m1).
Reactions were run and analyzed using 7900 HT fast RT-PCR (Applied Biosystems, Foster City, CA).
When using the Taqman (Life Technologies, Carlsbad, CA), the following primers were used: p21 (00355782_m1) and Bax (00180269_m1).
Reactions were run and analyzed using 7900 HT fast RT-PCR (Applied Biosystems, Foster City, CA).
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