AllPrep DNA/RNA Mini Kit

RNA isolation / purification Tissue - Human Retina

Experiment
RNA isolation / purification Tissue - Human Retina
Product
AllPrep DNA/RNA Mini Kit from Qiagen
Manufacturer
Qiagen

Protocol tips

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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Discussion

Discussion

4 years ago

Author: Paul G. Macon United States

RNA isolation from tissue

How do I extract RNA from animal tissue without using liquid nitrogen? I tried the RNA extraction by using the TRIzol reagent and I homogenize the tissue using polytron homogenizer at room temperature for 30secs is this correct?

Discussion

5 years ago

Author: Aaron Stege Netherlands

Problem in phase separation after using serum/plasma kit

I used a serum/plasma kit for my serum samples. After the phase separation the samples should have 3 phases: a colourless aqueous phase, a white interphase and a red organic phase. However, in some of my samples there was no aqueous phase unless I wait for an extended period of time. How can I circumvent this problem?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing RNA isolation / purification Tissue - Human Retina using AllPrep DNA/RNA Mini Kit from Qiagen.

Paper title
Complete Transcriptome Profiling of Normal and Age-Related Macular Degeneration Eye Tissues Reveals Dysregulation of Anti-Sense Transcription
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Manufacturer protocol

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

Download PDF Download manufacturer protocol

Videos

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