rna-isolation-purification-cells-primary-mouse-cortical-neurons

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Get tips on using pMT/BiP/V5-His A, B, & C Drosophila Expression Vectors to perform Protein expression and purification Insect cells - S2 HER2

Products Thermo Fisher Scientific pMT/BiP/V5-His A, B, & C Drosophila Expression Vectors

Get tips on using M-PER™ Mammalian Protein Extraction Reagent to perform Protein isolation Mammalian cells - SK-N-BE(2)-C

Products Thermo Fisher Scientific M-PER™ Mammalian Protein Extraction Reagent

RNA siRNA / miRNA gene silencing Rat Glial cells C/EBP‐β

RNA siRNA / miRNA gene silencing Rat Retinal stem cells Brn-3b

The formation of DNA from an RNA template using reverse transcription leads to the formation of double-stranded complementary DNA or cDNA. The challenges with this process include 1. Maintaining the integrity of RNA, 2. Hairpin loops or other secondary structures formed by single-stranded RNA can also affect cDNA synthesis, and 3. DNA-RNA hybrids, which may result when the first strand of cDNA is formed. For the first challenge, using workflows that involve proper isolation and storage of RNA, and maintaining a nuclease-free environment helps obtain RNA with ideal 260/230 ratios. Using a reverse transcriptase that can tolerate high temperatures (50-55oC), overcomes obstacles imposed by secondary RNA structures. Finally, RNase H has the ability to hydrolyze RNA before the formation of a second cDNA strand. It is important to ensure that RNase H activity is optimal because higher RNase H activity leads to premature degradation of the RNA template. Many reverse transcriptases offer built-in RNase H activity.

RNA cDNA synthesis Tissue

The formation of DNA from an RNA template using reverse transcription leads to the formation of double-stranded complementary DNA or cDNA. The challenges with this process include 1. Maintaining the integrity of RNA, 2. Hairpin loops or other secondary structures formed by single-stranded RNA can also affect cDNA synthesis, and 3. DNA-RNA hybrids, which may result when the first strand of cDNA is formed. For the first challenge, using workflows that involve proper isolation and storage of RNA, and maintaining a nuclease-free environment helps obtain RNA with ideal 260/230 ratios. Using a reverse transcriptase that can tolerate high temperatures (50-55oC), overcomes obstacles imposed by secondary RNA structures. Finally, RNase H has the ability to hydrolyze RNA before the formation of a second cDNA strand. It is important to ensure that RNase H activity is optimal because higher RNase H activity leads to premature degradation of the RNA template. Many reverse transcriptases offer built-in RNase H activity.

RNA cDNA synthesis Yeast

Get tips on using Senescence Cells Histochemical Staining Kit to perform Reporter gene assay β-galactosidase substrates - adipose stem cells

Products Sigma-Aldrich Senescence Cells Histochemical Staining Kit

Get tips on using Senescence Cells Histochemical Staining Kit to perform Reporter gene assay β-galactosidase substrates - human MSCs (mesenchymal stem cells)

Products Sigma-Aldrich Senescence Cells Histochemical Staining Kit

An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

Cellular assays Live / Dead assay mammalian cells HepaRG human hepatoma

An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

Cellular assays Live / Dead assay mammalian cells human fibroblast tissue

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