Ssofast green qpcr kit

Manufactured by Bio-Rad

Sourced in United States

The SsoFast Green qPCR kit is a real-time PCR reagent designed for high-performance quantitative PCR (qPCR) analysis. It contains a fast-cycling, hot-start DNA polymerase and an intercalating dye for detection of double-stranded DNA amplification.

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SsoFast qPCR kit (3 citations) QPCR kits (2 citations) SsoFast Eva Green qPCR kit (2 citations)

5 protocols using ssofast green qpcr kit

Quantification of miR-125a/b and FLT3-ITD in AML

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Peripheral blood samples were collected from 14 AML patients with informed consents at Queen Mary Hospital, Hong Kong. Total peripheral blood mononuclear cells were purified according to a previous protocol.²¹ The samples were confirmed to have at least 38% of leukaemia blasts. Total RNA extraction was carried out using TRIzol (ThermoFisher) based on the manufacturer's instructions. The quality and quantity of RNA samples were evaluated by NanoDrop analysis (ThermoFisher) and agarose gel electrophoresis. RNA samples were reverse transcribed into cDNAs using a reverse transcription kit (ThermoFisher) according to the manufacturer's protocol. Taqman® miRNA assays (ThermoFisher) were used to quantify the levels of miR‐125a and miR‐125b, and U6b RNA was used as the internal control. Ssofast Green qPCR kit (Bio‐Rad) was used to quantify the levels of FLT3‐ITD mRNAs, relative to the level of GAPDH. VIIA(TM) 7 System (Applied Biosystems, USA) was used to process all qPCR reactions.

Chen H., Jayasinghe M.K., Yeo E.Y., Wu Z., Pirisinu M., Usman W.M., Pham T.T., Lim K.W., Tran N.V., Leung A.Y., Du X., Zhang Q., Phan A.T, & Le M.T. (2022). CD33‐targeting extracellular vesicles deliver antisense oligonucleotides against FLT3‐ITD and miR‐125b for specific treatment of acute myeloid leukaemia. Cell Proliferation, 55(9), e13255.

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Quantification of mRNA and miRNA Levels

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Total RNA was extracted from cells or tissues using TRIzol (Thermo Fisher Scientific) according to the manufacturer's manuals. RNA was converted to cDNA using a high capacity cDNA reverse transcription kit (Thermo Fisher Scientific) following the manufacturer's protocol. mRNA levels were quantified using Ssofast^® Green qPCR kit (Bio‐Rad), normalized to GAPDH (for primer sequences, see Supplementary Table S1). miRNA levels were quantified using Taqman^® miRNA qPCR kit (Thermo Fisher Scientific), normalized to snoRNA234 (mouse) or U6B snRNA (human). All qPCR reactions were performed using a CFX96 Touch™ Real‐Time PCR Detection System (Bio‐Rad) or a QuantStudio 6 Flex Real‐Time PCR System (Life Technologies).

Peng B., Nguyen T.M., Jayasinghe M.K., Gao C., Pham T.T., Vu L.T., Yeo E.Y., Yap G., Wang L., Goh B.C., Tam W.L., Luo D, & Le M.T. (2022). Robust delivery of RIG‐I agonists using extracellular vesicles for anti‐cancer immunotherapy. Journal of Extracellular Vesicles, 11(4), e12187.

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Quantitative miRNA and mRNA Analysis

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RNA was diluted to 50 µg/µL for reverse transcription (RT). A total of 100 µg total RNA were used for miRNA RT and 500 µg total RNA was used for mRNA RT, with a high capacity cDNA RT kit (Life Technologies). qPCR was conducted using the TaqMan miRNA qPCR kit (Thermo Fisher Scientific) or Ssofast Green qPCR kit (BioRad) for miRNA and mRNA quantification, respectively, in a CFX-96 qPCR machine (Bio-Rad, USA). Cellular miRNA levels were normalized to snoRNA234 (mouse) or U6B snRNA (human). mRNA levels were normalized to GAPDH (for primer sequences, see Supplementary Table 1).

Vu L.T., Peng B., Zhang D.X., Ma V., Mathey-Andrews C.A., Lam C.K., Kiomourtzis T., Jin J., McReynolds L., Huang L., Grimson A., Cho W.C., Lieberman J, & Le M.T. (2019). Tumor-secreted extracellular vesicles promote the activation of cancer-associated fibroblasts via the transfer of microRNA-125b. Journal of Extracellular Vesicles, 8(1), 1599680.

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RNA Extraction and qPCR Analysis

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TRIzol (Thermo Fisher, USA) was used for RNA extraction, according to the manufacturer's protocol. We diluted the stock RNA to 50 ng/μl for reverse transcription. A total of 500 ng total RNA was applied for reverse transcription of mRNA, with a High‐Capacity cDNA Reverse Transcription Kit (Thermo Fisher, USA). Quantitative PCR (qPCR) was performed using a Ssofast Green qPCR kit (BioRad, USA) for mRNA quantification in a CFX‐96 qPCR machine (BioRad, USA) or QuantStudio 6 Flex Real Time PCR system (Applied Biosystems, USA). mRNA levels were normalized to GAPDH or ATCB mRNA levels. Primer design was facilitated using PrimerBank (Amin et al., 2017 ) (10.1093/nar/gkr1013) and Primer‐BLAST (Wang et al., 2011 (link)). We attached the primer sequences in Table S6.

Zhang D.X., Dang X.T., Vu L.T., Lim C.M., Yeo E.Y., Lam B.W., Leong S.M., Omar N., Putti T.C., Yeh Y.C., Ma V., Luo J., Cho W.C., Chen G., Lee V.K., Grimson A, & Le M.T. (2022). αvβ1 integrin is enriched in extracellular vesicles of metastatic breast cancer cells: A mechanism mediated by galectin‐3. Journal of Extracellular Vesicles, 11(8), e12234.

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RNA Extraction and qPCR Analysis of Macrophages

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Total RNA was extracted from macrophages using TRIzol™ Reagent (ThermoFisher Scientific) according to the manufacturer's instructions. The RNA was converted to cDNA using a high‐capacity cDNA reverse transcription kit (ThermoFisher Scientific) and quantified with the Ssofast® Green qPCR kit (Bio‐Rad), normalized to the expression of GAPDH, according to the manufacturers’ instructions. All the qPCR reactions were performed using a QuantStudio 6 Flex Real‐Time PCR system (Life Technologies).

Pham T.T., Le A.H., Dang C.P., Chong S.Y., Do D.V., Peng B., Jayasinghe M.K., Ong H.B., Hoang D.V., Louise R.A., Loh Y., Hou H.W., Wang J, & Le M.T. (2023). Endocytosis of red blood cell extracellular vesicles by macrophages leads to cytoplasmic heme release and prevents foam cell formation in atherosclerosis. Journal of Extracellular Vesicles, 12(8), 12354.

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