To extract RNA from bone, liquid nitrogen was continuously and rapidly added to grind down alveolar bone until a powder was generated. Also, spleens were removed under sterile conditions and lymphocytes isolated to extract RNA. Total RNA was extracted from bone and cell samples using Trizol reagent (AG21102, Accurate Biotechnology Co., Ltd. China). cDNAs were synthesized using the Evo M-MLV RT Reverse Transcription kit II (AG11711, Accurate Biotechnology Co., Ltd.) according to manufacturer’s instructions. Using qRT-PCR instrumentation (LightCycler® 96 SW 1.1; Roche Ltd., Switzerland), qRT-PCR was performed using the SYBR Green Pro Taq HS premixed qRT-PCR kit (AG11701, Accurate Biotechnology). Treg cell-related factors (transforming Growth Factor-β (TGF-β) and FOXP3) and Th17 cell-related factors (retinoic acid-related orphan receptor γ (ROR-γt) and IL-17A) were analyzed. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control, and results were presented as relative gene expression. Fold-change in gene expression was calculated relative to controls using the 2-∆∆Cq method in GraphPad Prism software (GraphPad Inc. United States). Primer sequences are shown (Table 1 ).
Evo m mlv rt reverse transcription kit 2
Manufactured by Accurate Biology
The Evo M-MLV RT Reverse Transcription kit II is a molecular biology tool used to convert RNA into complementary DNA (cDNA). It contains the necessary components, including reverse transcriptase enzyme, buffer, and other reagents, to perform this process.
Automatically generated - may contain errors
2 protocols using evo m mlv rt reverse transcription kit 2
1
Extracting and Analyzing RNA from Bone and Immune Cells
2
Quantitative RT-PCR Analysis of Cell Lines
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CAL27
and SCC15 cell RNA was extracted from the cells using Trizol
reagent (AG21102, Accurate Biotechnology Co., Ltd., China). cDNA was
synthesized using the Evo M-MLV RT Reverse Transcription kit II (AG11711,
Accurate Biotechnology). QPCR was performed using the SYBR Green Pro
Taq HS premixed qPCR kit (AG11701, Accurate Biotechnology) in an RT
fluorescence quantitative PCR system (Light Cycler 96 SW 1.1, Roche
Ltd, Switzerland). The parameters required for denaturation, annealing,
and extension were as follows: 95 °C for 30 s, 45 cycles at 95
°C for 5 s, and 60 °C for 20 s. The primer sequences are
shown inTable 1 . All
data were normalized to GAPDH expression. Quantification of the qRT-PCR
results was performed by the 2–ΔΔCT method.
and SCC15 cell RNA was extracted from the cells using Trizol
reagent (AG21102, Accurate Biotechnology Co., Ltd., China). cDNA was
synthesized using the Evo M-MLV RT Reverse Transcription kit II (AG11711,
Accurate Biotechnology). QPCR was performed using the SYBR Green Pro
Taq HS premixed qPCR kit (AG11701, Accurate Biotechnology) in an RT
fluorescence quantitative PCR system (Light Cycler 96 SW 1.1, Roche
Ltd, Switzerland). The parameters required for denaturation, annealing,
and extension were as follows: 95 °C for 30 s, 45 cycles at 95
°C for 5 s, and 60 °C for 20 s. The primer sequences are
shown in
data were normalized to GAPDH expression. Quantification of the qRT-PCR
results was performed by the 2–ΔΔCT method.
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