hASCs were seeded onto 6-well culture plates (1 × 105 cells per well), cultured for 24 h at 37°C in DMEM containing 1% FBS with or without LMWH. Total RNA was extracted from the cultured hASCs. Following RNA extraction with an RNeasy Mini Kit (Qiagen Ltd., Manchester, UK), cDNA was synthesized using an ExScript RT kit (Takara, Shiga, Japan), and amplification was performed on a ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Tokyo, Japan) according to the manufacturer’s instructions. The primer sequences for CXC chemokine receptor 7 (CXCR7), CXC chemokine receptor 4 (CXCR4), HGF, CXCL12, phosphatidylinositol-3-kinase (PI3K), AKT, and glyceraldehyde 6-phosphate dehydrogenase (GAPDH) as a housekeeping gene are listed in Supplementary Table 1
Exscript rt kit
Manufactured by Takara Bio
Sourced in Japan
The ExScript RT kit is a reverse transcription kit designed for the synthesis of complementary DNA (cDNA) from RNA templates. The kit includes all the necessary components for the reverse transcription reaction, including a thermostable reverse transcriptase enzyme and optimized reaction buffers.
Automatically generated - may contain errors
Lab products found in correlation
6 protocols using exscript rt kit
1
Expression Profiling of hASCs with LMWH
2
qPCR Analysis of Alfalfa Transcripts
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Total RNA from alfalfa seedlings was isolated using the Ultra-Pure RNA Kit (cwbiotech, Beijing, China) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using the ExScript RT kit (Takara Bio. Beijing, China). Quantitative PCR (qPCR) was performed on a Realplex 4 Master Cycler using a gene-specific primer (Table S1 ) using Super Real PreMix Plus with Syber Green 1 (TIANGEN Biotech., Beijing, China). Relative gene expression was assessed using a comparative cycle threshold method [40 (link)]. The relative gene expression was calculated as follows: 2−ΔΔCT (ΔCT = CT, gene of interest-CT versus actin) as described by [41 (link)], where the actin gene was used as a reference. A mixture cDNA of 30 leaf samples was used to perform the qPCR reactions to determine the primer efficiency as described previously [42 (link)]. All primer amplification efficiencies were between 99% and 104% (Table S1 ). Three complete biological and technical replicates were performed.
3
Validating Differential Gene Expression in Maize
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Real-time PCR (qPCR) was used to validate the differentially abundant genes in different background of maize varieties. Total RNA from maize leaves was isolated using Ultra-Pure RNA Kit (cwbiotech), and complementary DNA was synthesized with ExScript RT Kit (Takara). The amplification reaction conditions were as follows: 95 °C for 3 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The primer is listed in Table S1. Actin gene was used as internal control. The relative gene expression level was calculated based on 2−ΔΔCT as described previously37 (link). Three biological replicates were performed.
4
Quantifying Murine Macrophage Gene Expression
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Total RNA was extracted from cultured murine macrophages. Following RNA extraction using the RNeasy Mini Kit (Qiagen Ltd., Manchester, UK), cDNA was synthesized using an ExScript RT kit (Takara, Shiga, Japan), and gene amplification was performed using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Tokyo, Japan) according to the manufacturer’s instructions. The primer sequences for CD64, CD163, IL-12, IL-10, and GAPDH (housekeeping gene) are summarized in Supplementary Table S1 . The relative mRNA expression of each target gene was calculated using the comparative CT method. The experiments were independently repeated four times for each sample and performed in triplicate.
5
LMWH Modulates Gene Expression in mASCs
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Total RNA was extracted from mASCs cultured in DMEM/F-12 containing 2% FBS and 1.0% Pen-Strep supplemented with LMWH at 0, 1, 10, or 100 μg/mL. After RNA extraction with an RNeasy mini kit (Qiagen Ltd., Manchester, UK), cDNA was synthesised using an ExScript RT kit (Takara, Shiga, Japan) and amplification was performed in a Sequence Detection System 7000 (Applied Biosystems) according to the manufacturer’s instructions. The primer sequences for stromal derived factor-1 (SDF-1), C-X-C chemokine receptor (CXCR) type 4 (CXCR-4), CXCR type 7 (CXCR-7), and glyceraldehyde 6-phosphate dehydrogenase (GAPDH) are summarised in Table 1 . Each target gene expression was normalised to the housekeeping gene expression. The experiments were repeated in triplicate, and results were averaged.
Primers used in the RT-qPCR analyses in vitro
| Gene | Forward | Reverse |
|---|---|---|
| CXCR-4 | 5′-TCATCAAGCAAGGGTGTGAG-3′ | 5′-GGCTCCAAGGAAAGCATAGA-3′ |
| CXCR-7 | 5′-AGAAGATGGTACGCCGTGTCG-3′ | 5′-TCTTCCGGCTGCTGTGCTTCTC-3′ |
| SDF-1 | 5′-AGCCATGTTGCCAGAGCCAACG-3′ | 5′-CACACACACACCTGGTCCTCATGG-3′ |
| GAPDH | 5′-ACAATGAATACGGCTACAG-3′ | 5′-GGTCCAGGGTTTCTTACT-3′ |
6
Metabolite Changes in Brassica under Low Light
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To perform the changes of metabolites induced by low light, ~2gBrassicastalkswere sampled from 3 days shading treatments and control, and were extracted using pre-cooled metal beads in a 2 mL Eppendorf tube, at 30 Hz for 3 min. We then dissolvedthe extracted powder with 1.3 mL methanol/chloroform, followed by incubation of the powder at -20 °C for 4 h, and centrifuged the mixture at 2,000 g at 4 o C for 10 min. The mixture was then ltered using 0.43 μm organic phase medium (GE Healthcare, 6789-0404), and the 10 μL mixture was loaded to HPLC for analysis. Mobile phase: 15% buffer A (95% H 2 O and 5% ACN, pH = 9.5) + 85% buffer B (100% ACN), 0.2 ml.min -1 . Three biological replicates were conducted for non-targeted metabolic measurements. q-RT-PCR analysis Total RNA was isolated from XH1 stalks with Ultra-Pure RNA Kit (cwbiotech) following the instructions of manufacturer, and cDNA was synthesized using ExScript RT kit (Takara). We performed q-RT-PCR on a Realplex 4 Master Cycler using Syber Green kit (TIANGEN). Calculation of relative gene expression was followed as reported previously (Livak and Schmittgen 2001) , where actin1 gene was used as a reference. At least three biological replicates were performed.
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