Computational protocol: Innate Immune Response and Off-Target Mis-splicing Are Common Morpholino-Induced Side Effects in Xenopus

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Protocol publication

[…] Approximately 750 ng total RNA was reverse transcribed with 40 U RNase H minus and point-mutant M-MLV reverse transcriptase (Promega, Cat#M3681), 500 μM of each dNTP and 10 μM random hexamers in a 10 μl reaction following this temperature regime: 15 mins at 25°C, 15 mins at 37°C, 45 mins at 55°C and 15 mins at 85°C. The RT reaction was subsequently diluted to 60 to 100 μl with molecular grade water for qPCR. 2 μl of the diluted RT reactions were amplified in technical duplicates with SYBR Green I master mix (Roche, Cat#04707516001) on a Light Cycler 480 II (Roche) cycling 55-times between 94, 60 and 72°C with each temperature step running for 10 secs and switching at +4.8°C/sec and -2.5°C/sec. At the end qPCR reactions were heated from 65 to 97°C with a gradual increase of 0.11°C/sec (melting curve) to ensure only fluorescence was collected from one specific amplicon. B was based on absolute quantification while all other RT-qPCR results were normalized to odc1 and uninjected embryos using the 2−ΔΔCt method (). The threshold cycle (Ct) was derived from the maximum acceleration of SYBR fluorescence (second derivative maximum method). The PCR primers were designed to hybridize at ∼60°C (Tm) and to generate 75 to 125 bp amplicons using Primer3 (). […]

Pipeline specifications

Software tools ddCt, Primer3
Application qPCR
Diseases Down Syndrome