piwik-script

Deutsch Intern
    Core Unit Systems Medicine

    Help & FAQ

    How should I send my samples?

    • Always on dry ice!

    How much RNA will I need?

    • Total RNA for rRNA-Depletion: ~ 1 µg
    • Total RNA for mRNA enrichment with Oligo-dT-capture beads: ~ 100 ng (down to 10 ng for high quality RNA)
    • Total RNA for ultra low input samples: ~ 10 pg - 10 ng (see also SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing)

    For which organisms do you have rRNA depletion solutions?

    • Illumina TruSeq Stranded Total RNA Gold:  human, mouse, rat
    • Illumina TruSeq Stranded Total RNA with Ribo-Zero Globin (Globin mRNA incl. rRNA Depletion):  human, mouse, rat
    • siTOOLs Biotech Pan-Prokaryote riboPOOL:  Bacteria / Archaea

    Tips for working with RNA:

    • Wear "fresh" disposable gloves
    • Clean bench and instruments (pipettes, ...) with cleaning solutions, such as RNaseZAP, RNase Away, ...
    • Spatial separation of the workplace for RNA work from workplaces working with RNase
    • Use sterile filter pipette tips
    • Use disposable sterile plastic as well as DEPC-treated reagents and glassware
    • follow protocols with short incubation times on ice and keep tubes closed whenever possible
    • if possible use of nuclease-free water (DEPC-treated or DNase / RNase-free)
    • Storage of RNA at -80 ° C in nuclease-free water (up to 1 year)
    • Link:  “The Do's and Don'ts of Working with RNA

    How much DNA is needed?

    • approx. 50 ng eukaryotic genomic DNA in 2 µl for WES
    • approx. 1 ng genomic DNA (e.g. bacterial DNA) in 1 µl for tagmentation (transposase-based enzymatic DNA fragmentation (approx. every 300 bp) with adapter ligation for PCR amplification)

    Tips for stabilizing RNA:

    • for cells: resuspend pelleted cells (approx. 0.5-1x10 ^ 6) in 0.3 mL RNAprotect Cell Reagent (e.g. QIAGEN Cat No. 76526) and store the suspension for 1 day at 4°C and then at -80°C until RNA isolation
    • for tissue: prepare pieces of tissue of approx. 0.5 cm x 0.5 cm x 0.5 cm. Transfer the tissue piece into a 2 ml microcentrifugation tube filled with 2 ml RNAlater (e.g. ThermoFisher QIAGEN Cat No. AM7020). Store the liquid for 1 day at 4°C and then at -80°C until RNA isolation

    Tips for stabilizing DNA:

    • for isolated DNA: add 25 μl DNAstable Plus (e.g. Biomatrica Cat No. 53091-016) to 100 μl DNA (0.001 to 250 µg) and store the liquid for up to 12 months at room temperature or 4 ° C
    • for blood samples: mix 200 µL blood with the DNAstable Blood matrix (e.g. Biomatrica Cat No. 93027-027) in a microcentrifugation tube, dry the matrix and store the dried matrix for up to 12 months at room temperature or -20°C
    • for cells and tissues: transfer approx. 10 mg tissue in 100 µL DNAgard tissue or resuspend pelleted cells (approx. 0.5-1x10^6) in 100 µL DNAgard tissue (e.g. Biomatrica Cat No. 62001-046). Store cells or tissues for up to 6 months at room temperature or -20°C.