AllPrep DNA/RNA Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the nucleic acids contained in the sample. - Different samples require different methods to achieve complete disruption. - Incomplete disruption results in significantly reduced nucleic acid yields. - Overloading the spin columns significantly reduces nucleic acid yields.	- Homogenizing the material is necessary to redice the viscosity of the lysates caused by cell disruption. - Incomplete homogenization results in inefficient binding of DNA and RNA and therefore significantly reduced yield and purity of nucleic acids. - Excessive homogenization, on the other hand, results in shorter genomic DNA fragments. - The centrifugation temperature should be 20–25ºC. - Warm the lysate to 37ºC before transferring it to the AllPrep DNA spin column.

Upstream tips
- Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the nucleic acids contained in the sample. - Different samples require different methods to achieve complete disruption. - Incomplete disruption results in significantly reduced nucleic acid yields. - Overloading the spin columns significantly reduces nucleic acid yields.
Protocol tips
- Homogenizing the material is necessary to redice the viscosity of the lysates caused by cell disruption. - Incomplete homogenization results in inefficient binding of DNA and RNA and therefore significantly reduced yield and purity of nucleic acids. - Excessive homogenization, on the other hand, results in shorter genomic DNA fragments. - The centrifugation temperature should be 20–25ºC. - Warm the lysate to 37ºC before transferring it to the AllPrep DNA spin column.

Publication protocol

Eight normal and eight AMD donor eyes produced 16 PR and 16 PRCS samples resulting in 32 RNA assays. These were prepared using the AllPrep DNA/RNA Mini kit (Qiagen). The RNA quality was determined using R6K Screen Tape on a 2200 Tape Station (Agilent, Santa Clara, CA, USA) and was quantified using Qbit-BR (Broad Range) assay kit on a Qbit 2.0 Fluorometer (Life Technologies) following manufacturers instruction. Only RNA with integrity number (RIN) value of >8.5 was used for preparing sequencing libraries. To sequence the transcriptome, library preparation and sequencing was done using the TruSeq Stranded total RNA with RiboZero Gold kit (Illumina, CA) protocol with a total RNA (800 ng) as the starting material. A total of 32 libraries were prepared with unique barcode sets and their quality was determined using Agilent DNA1000 chip following manufactures protocol. All the DNA libraries with mean peak size of 260 bp were processed for sequencing. The libraries were sequenced on an Illumia HiSeq. 2000 machine following manufacturers protocols.

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Manufacturer protocol

Download the product protocol from Qiagen for AllPrep DNA/RNA Mini Kit below.

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