PAXgene Bone Marrow RNA Kit

The supernatants resulting from the first two spin steps of the PAXgene Bone Marrow RNA Kit purification procedure may still contain biologically hazardous material. We recommend classifying the tube-processed supernatant of the kit as both a biological and chemical waste. Supernatants can be decontaminated by autoclaving and disposed of as chemical waste.  Disposal should be done in accordance with local, state and federal regulations.

CAUTION: Do not add bleach or acidic solutions directly to the sample preparation waste.

The PAXgene Bone Marrow RNA System was developed for intracellular RNA isolation from human bone marrow samples. Bone marrow samples from other species or from blood were not tested.

Yes. We have tested the PAXgene Bone Marrow RNA System with bone marrow samples containing very high numbers of leukocytes. Bone marrow samples with higher leukocyte numbers are indicated by a highly viscous solution after addition of buffers BMR1 and BMR2 followed by incubation. These challenging samples are further diluted according to descriptions given in the protocol and do not lead to problems in the extraction procedure.

Yes. You may dispose of the waste product from the PAXgene Bone Marrow RNA Tube with a 5% sodium hypochlorite bleach solution, in a 1:9 ratio (1 part bleach : 9 parts tube contents).

The PAXgene Bone Marrow RNA Tubes should be frozen at –20°C and –70°C in a wire rack, not in an expanded polystyrene foam tray.

If the PAXgene Bone Marrow RNA Tubes are to be stored at temperatures lower than –20ºC, freeze them first for 24 h at –20ºC, then transfer them to –70ºC.

Currently available data shows stabilization of cellular RNA for several months at –20°C or –70°C. For more information from ongoing studies evaluating stability for longer time periods, please contact QIAGEN Technical Services.

 

 

Bone marrow samples are extremely heterogeneous, consisting of varying amounts of cells, tissue, and solid matter. RNA yields are highly donor-dependent and can vary greatly from sample to sample as well as between replicates from the same donor due to sample-intrinsic heterogeneity.

In a study of 295 bone marrow samples, yields of RNA isolated from 2 mL human bone marrow were between 2.0 and 44.7 µg (5-95% quantiles, mean = 15.8 µg, median = 11.0 µg).

Yes. The PAXgene Bone Marrow RNA System purifies RNA from whole bone marrow. Therefore it will also purify RNA from other cells, e.g. reticulocytes and RBCs. Array analyses have shown higher globin mRNA levels for RNA isolated from whole bone marrow compared to RNA isolated from mononuclear cells.

No. The 260/230 nm absorbance ratio is not specified for the PAXgene Bone Marrow RNA extraction kit. In contrast to a reduced 260/280 ratio, which results from a protein contamination of the eluted RNA and can lead to inhibitory effects in down stream applications, there is no negative impact of a reduced 260/230 ratio on any down stream application reported to date and therefore it is not necessary to specify this.

In addition, because the elution buffer contains very low concentrations of salt that absorb at wavelengths between 220 and 230 nm, it is necessary to zero the photometer properly. Do this by preparing a solution for zeroing the photometer which contains a similar salt concentration as the sample to be measured by using the same volumes. Add the same volume of Buffer BMR5 as the volume of RNA sample to be diluted into a fresh volume of the buffer in which you will measure the absorption (we recommend 10 mM Tris Cl pH 7.5 buffer for this purpose).

The PAXgene Bone Marrow RNA kit elution buffer (Buffer BMR5) is a proprietary low-salt buffer. In combination with the final heat denaturation step, it leads to optimal performance of the isolated RNA in RT-reactions. There is no interference with downstream applications reported to date.

No. The PAXgene Bone Marrow RNA System was developed for intracellular RNA isolation from human bone marrow samples. PreAnalytiX offers a dedicated collection and RNA extraction system for human blood (see the PAXgene Blood RNA System). QIAGEN also offers RNA stabilization and isolation systems for saliva and cultured cells. Please contact QIAGEN technical service for more information or visit www.qiagen.com.

No. Transferring bone marrow volumes different than 2mL into the PAXgene Bone Marrow RNA Tube is not recommended. The tube and its contents were optimized for performance with 2 mL of bone marrow. The use of other volumes is not recommended.

No. The PAXgene Bone Marrow RNA System has been optimized only for cellular RNA only.