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View additional product information for MagMAX™ mirVana™ Total RNA Isolation Kit - FAQs (A27828)
41 product FAQs found
Yes, you can. The DNA Binding Beads for MagMAX-96 DNA Multi-Sample Kit (Cat. No. 4489112) are the same as that used in A27828.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
We have tested extraction from as low as 1,000 cells using cultured HeLa, Jurkat, and HepG2 cells.
Detection of somatic variants with the Oncomine Myeloid Research Assay GX has been verified down to 5% allele frequency.
Here is a list of kits that are compatible with Oncomine Myeloid Research Assay GX:
Nucleic acid isolation - RNA samples
- MagMAX mirVana Total RNA Isolation Kit, Cat. No. A27828
- PureLink Total RNA Blood Kit, Cat. No. K156001
- RNaseZap RNase Decontamination Solution, Cat. No. AM9780, AM9782, AM9784
Nucleic acid isolation - DNA samples
- MagMAX DNA Multi-Sample Ultra 2.0 Kit, Cat. No. A36570
- PureLink Genomic DNA Mini Kit, Cat. No. K1820-01
The recommended minimum sample input is 27.75 ng of DNA (1.11 ng/µL minimum concentration) and 14.25 ng of RNA (0.95 ng/µL minimum concentration).
The recommended controls are:
DNA Controls:
- Seraseq® Myeloid Mutation DNA Mix, Cat. No. 0710-0408 (SeraCare®)
- Myeloid DNA Reference Standard, Cat. No. HD829 (Horizon™)
- AcroMetrix™ Oncology Hotspot Control, Cat. No. 969056
RNA Controls:
- Universal Human Reference RNA, Cat. No. 740000 (Agilent™)
- Seraseq® Myeloid Fusion RNA Mix, Cat. No. 0710-0407 (SeraCare®)
Multiplex sequencing of up to 8 Oncomine Myeloid Research Assay GX samples (8 DNA+ 8 RNA) per lane on a GX5 Chip can be performed in a single run. There are 4 lanes on the GX5 Chip, so 32 total samples (32 DNA+ 32 RNA) can be run on one GX5 Chip (8 samples per run).
The Oncomine Myeloid Research Assay GX (Cat. No. A47857) includes the 2-pool DNA panel, the 1-pool RNA panel, Genexus Strip 1, and Genexus Strip 2-AS. The DNA panel is provided in two packs of 8 tubes (4 tubes of Myeloid DNA Pool 1 and 4 tubes of Myeloid DNA Pool 2 per pack). The RNA panel is provided in one pack of 8 tubes (Myeloid RNA Pool 1). Each primer pool in the panel is provided in pairs of tubes, where each tube pair contains one tube with primers in position 1 and one empty uncapped tube in position 2. Three 8-strip packs of the Genexus Strip 1 and Genexus Strip 2-AS (24 total of each strip) are provided with each kit.
The contents of each Oncomine Myeloid Research Assay GX kit are sufficient for up to 32 samples (32 x 3‑pool reactions: 32 x 2‑pool DNA reactions and 32 x 1‑pool RNA reactions).
The Oncomine Myeloid Research Assay GX includes 3 pools of Ion AmpliSeq oligonucleotide primers (a 2-pool DNA panel and a 1-pool RNA panel).
The Oncomine Myeloid Research Assay GX targets key genes and fusions associated with major myeloid disorders, including acute myeloid leukemia (AML), myeloid dysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML). The DNA panel comprises 40 key genes while the RNA panel includes a broad fusion panel of 29 driver genes, enabling detection over 600 unique fusion isotypes. See flyer for gene targets (https://assets.thermofisher.com/TFS-Assets/CSD/Flyers/Oncomine_myeloid_assay_gx_flyer.pdf).
The Oncomine Myeloid Research Assay GX is a comprehensive targeted next-generation sequencing (NGS) assay designed for sensitive detection of myeloid disorder-associated DNA mutations and fusion transcripts in blood and bone marrow samples.
Some bead carryover is common and has not been documented to affect downstream assays. The most common cause of bead carryover is too much input sample. The best way to determine if the sample amount is the cause of the issue is to run PBS with Xeno (artificial DNA/RNA) in the lysis buffer and then check if there is still bead carryover. If that is not the cause, the initial sample may require special processing, such as bead beating or liquification. If there is still bead carryover without the original sample, and in particular, if the carryover is present in the same wells for multiple runs, then this may be an instrument alignment issue.
Plastic consumables need to be purchased separately in order to use the MagMAX reagents on the MagMAX Express-96 or a KingFisher instrument. The particular consumables and the amount required will vary by kit and protocol so please check the user guide for the particular MagMAX kit to be sure that all required plastic consumables are available prior to sample processing.
The best place to download KingFisher Flex and KingFisher Duo Prime protocols for a particular MagMAX kit is the relevant MagMAX kit product page. The Bindlt script protocol (.bdz) files can be found under the Documents>Product literature section.
Yes, reaction volumes for MagMAX kits can be scaled up. In general, the amount of MagMAX reagents should be scaled up proportionally for the increase in sample input amount. Please see the specific kit user guide for any recommendation. If none are included, then optimization may be necessary.
Addition of BME (2-mercaptoethanol) to the lysis buffer of MagMAX kits is fine. This can be beneficial with more RNase-rich samples like certain types of tissues.
In general, we recommend adding 0.3 µL BME per 100 µL of lysate.
Lysates made using the lysis buffer from the MagMAX mirVana Total RNA Isolation Kit can be stored at -20 to -80 degrees C for up to 4 days if 2-mercaptoethanol or 100 mM DTT is added to the lysis buffer prior to homogenization of the sample.
For tissues containing low to normal levels of cellular RNase (for example, brain, heart, or liver) we recommend using 20 µL of Lysis Binding Mix for 1 mg of tissue (1:20 ratio). For example, use 200 µL of Lysis Binding Mix for 10 mg of tissue.
For tissues containing high levels of cellular RNase (for example, spleen or pancreas), we recommend using 40 µL of Lysis Binding Mix for 1 mg of tissue (1:40 ratio). For example, use 400 µL of Lysis Binding Mix for 10 mg of tissue.
We recommend homogenizing for 30 s for full lysis. Our protocol recommends use of single sample polytron homogenizer, but you can also use bead-beating instrument with glass or zirconia beads. You may need to optimize bead-beating for your specific sample.
No, only certain MagMAX kits are compatible with Tempus/PAXgene stabilized blood samples. We recommend using only MagMAX kits that are designed for blood samples stored in the designated RNA blood tubes for the kit. For PAXgene tubes, the compatible kit is the MagMAX for Stabilized Blood Tubes RNA Isolation Kit, compatible with PAXgene Blood RNA Tubes (Cat. No. 4451894). For Tempus tubes the compatible kit is the MagMAX for Stabilized Blood Tubes RNA Isolation Kit, compatible with Tempus Blood RNA Tubes (Cat. No. 4451893).
Frozen serum/plasma samples can be used with the MagMAX mirVana Total RNA Isolation Kit, though we recommend fresh samples. We also recommend limiting freeze/thaw cycles to 1-2 cycles to prevent RNA degradation.
Frozen whole blood samples can be used with the MagMAX mirVana Total RNA Isolation Kit if the downstream application can use fragmented RNA (e.g., qRT-PCR, amplicon sequencing). However, collection of blood samples in Tempus Blood RNA Tubes (Cat. No. 4342792) or PAXgene tubes is recommended for long-term storage and when high-quality RNA is needed because freezing or prolonged storage at 4 degrees C (>24 hrs) results in RNA degradation.
The MagMAX mirVana Total RNA Isolation Kit user guide recommends up to 1x10^6 cells, but this can potentially be increased to 5x10^6 cells by increasing the lysis and binding reagents accordingly. This may not work well for all cell types, so we recommend testing control samples of the cell line of interest.
Yes, the input can be increased up to 150-200 µL for the automated protocol with all binding reagents kept proportionate.
The input for the serum/plasma protocol for the MagMAX mirVana Total RNA Isolation Kit can be increased to 200 µL of sample for the automated protocol if all binding reagents are increase proportionally.
The maximum input amount ranges from 2.5-10 mg of tissue depending on the tissue type. For example, spleen and thymus use a lower input amount. However, you can homogenize/lyse a larger piece and store the remaining lysate at -20 degrees C.
Further information regarding tissue input amounts can be found in the MagMAX mirVana Total RNA Isolation Kit (Tissue Samples) User Guide: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011132_A27828_magmax_rnaisolation_tissues_ug.pdf
Unfortunately, we do not have a script for using the MagMAX mirVana Total RNA Isolation Kit (Cat. No. A27828) on the MagMAX Express 96 instrument. However, we do have a free BindIt 4.0 software for converting KingFisher Flex scripts to MagMAX Express 96 scripts.
We do not sell the Proteinase K from the MagMAX mirVana Total RNA Isolation Kit as a standalone item. A different Proteinase K product (Cat. No. AM2548) can be purchased as a standalone item and can be used at 2.5X in the MagMAX mirVana protocol if you would like to add more Proteinase K to the procedure.
The MagMAX mirVana Total RNA Isolation Kit can be used to isolate RNA from exosomes. We first recommend isolating/concentrating exosomes with our Total Exosome Isolation Reagent into a volume of 100 µL (bring up with 1X PBS if needed) and then extracting the RNA using the serum/plasma protocol for MagMAX mirVana Total RNA Isolation Kit. The Proteinase K treatment will work well in place of the organic extraction that is normally used to lyse open the exosomes.
We have not tested any MagMAX kit with less than 50µL of whole blood. It may be possible to isolate RNA from 10µL of whole blood using the MagMAX mirVana Total RNA Isolation Kit (Cat. No. A27828) by bringing the sample volume up to 50 µL using 1X PBS and then processing the sample using the standard protocol.
The following are speed settings that we recommend using in conjunction with the MagMAX mirVana Total RNA Isolation Kit protocol:
- Speed 2 - 400 rpm
- Speed 4 - 700 rpm
- Speed 10 - 1150 rpm
We still recommend watching your samples to ensure that there is no spill over.
The TURBO DNase in the MagMAX mirVana Total RNA Isolation Kit is the same enzyme as the standalone TURBO DNase, but the concentrations of the enzyme differs and therefore it is not an exact equivalent. The TURBO DNase in the MagMAX mirVana Total RNA Isolation Kit is at 20 U/µL, whereas the standalone TURBO DNase is at 2 U/µL.
You can find scripts for using MagMAX mirVana Total RNA Isolation Kit with the MagMAX Express-96, KingFisher Flex, or KingFisher Duo instrument, on the product page (http://www.thermofisher.com/order/catalog/product/A27828).
No, we recommend preparing the Proteinase K Digestion Mix fresh each time.
Yes, you can, but we only recommend storing the lysed samples in Lysis Binding Mix at -20°C for up to 4 days. Samples should be thawed to room temperature before proceeding with the RNA purification.
Yes, it comes with one processing plate, two elution plates, and four plate covers to support manual processing. However, you will have to purchase deep-well plates, standard plates, and tip combs in addition to that if you wish to process on a KingFisher instrument.
Yes, the Applied Biosystems MagMAX mirVana Total RNA Isolation Kit (Cat. No. A27828) will isolate both small and large RNAs from a variety of samples (serum, plasma, blood, tissue, cells, urine).
No, manufactured protocols are locked for editing, but they can be renamed using the ‘save as' function and edited as a new version.
For nucleic acid extraction, we recommend using our MagMAX kits and MagJET kits. Other magnetic beads should work well as long as they are 0.5 to 10 µm in size. However, you will have to write your own KingFisher protocols using the Thermo Scientific BindIt software and validate/optimize the protocol on your own.
Find additional tips, troubleshooting help, and resources within our Automated Nucleic Acid Purification Support Center.
Yes, this can be done by sample splitting. Set the protocol so that the magnet will collect samples from multiple wells before continuing to the next step. Be sure not to overload.
Find additional tips, troubleshooting help, and resources within our Automated Nucleic Acid Purification Support Center.
Yes, reaction volumes can be scaled up, and all other volumes (i.e. wash volumes) will have to be increased by the same fold difference. It is generally not recommended to scale up reaction volumes more than 2-fold unless the plate's volume capacity will allow it.