RNA was isolated using TriPure reagent as described (Akerman et al., 2017) following manufacturer’s instructions. RNA quality was ascertained with a 2100 Agilent Bioanalyzer (RIN > 8). Ins2, INS, ActB and Ins2 intronic transcripts were measured using TaqMan (Applied Biosystems) probes, while other regions were quantified using oligonucleotide primers and SYBR green mastermix (Illumina). cDNA synthesis was carried out using Superscript III (Invitrogen) and real-time PCR was performed with the ABI 7300 Real Time PCR system using the Power SYBR Green reagent (Applied Biosystems). Serial dilutions of genomic DNA (5 data points) were used to establish a standard curve. SDS software (Applied Biosystems) was used to generate quantitative values based on the standard curve with arbitrary units. All mRNA levels were normalized to ActB or Hprt as indicated in Figure legends. Oligonucleotides and TaqMan probe sequences can be found in Table S3.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.