RNA isolation / purification Cells - primary human CD14+ monocytes

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.

Start discussion

Found 1 discussion for this experiment

Discussion

5 years ago

5 years ago by Ralf Friedmann Switzerland

I am having trouble using the QiageN RNAeasy mini kit for CD34+ cells. Any tips?

Comment View 1 comment

Share your thoughts or question with experts in your field by adding a discussion!

Found 3 matching solutions for this experiment

AllPrep DNA/RNA Mini Kit

Qiagen

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.

Manufacturer protocol Publication protocol 1 Relevant paper

RNeasy Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Protocol tips
- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Manufacturer protocol Publication protocol 1 Relevant paper

RNeasy Plus Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
		- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Downstream tips
- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Manufacturer protocol Publication protocol 1 Relevant paper

Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Fill out your contact details and receive price quotes in your Inbox