Allprep dna rna micro

Genomic DNA and total RNA were extracted simultaneously from fresh liver samples using the AllPrep DNA/RNA Micro (Qiagen, Dubai, United Arab Emirates). According to the manufacturer’s instructions, hepatic tissue samples were first lysed and homogenized in a buffer for inhibition of DNases and RNases to obtain intact DNA and RNA. The lysate was passed through an AllPrep DNA spin column to selectively and efficiently isolate DNA. The column was then washed and DNA was eluted. Ethanol was added to the flow-through from the AllPrep DNA spin column to allow proper binding conditions for RNA, and the sample was then applied to RNeasy MinElute spin column, where total RNA binds into the membrane and contaminants were effectively washed away. Finally, RNA was then eluted in water.

Messengers RNA (mRNA), small RNA, and DNA were simultaneously isolated using a combination of the following two kits: Qiagen All Prep DNA/RNA micro (cat. no. 80284, Hilden, Germany) and RNeasy Mini Kit (cat. no. 74104, Qiagen, Hilden, Germany). Briefly, the tubes with the collected outer cumulus cells were further filled to 350 µl of RLT Plus buffer and vortexed for one minute. The lysate was then loaded to an “All prep” DNA column and the bounded DNA was processed according to the kit instruction for DNA isolation. The flow-through containing the RNA was filled with 350 µl of 70% ethanol and loaded to an RNeasy mini-elute column and further processed according to kit instructions to isolate the long RNA. DNA and RNA were collected in 45 and 12 µL total volume and the yield was in the range of 200, 50, and 350 ng, respectively.