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RNA Sample Collection and Protection

RNAlater® Stabilization Solution is an aqueous tissue storage reagent that protects cellular RNA in intact, unfrozen tissue samples. RNAlater® Stabilization Solution eliminates the need to immediately process your tissue samples or to freeze them in liquid nitrogen for later processing. Read more about RNAlater® Stabilization Solution here.

RNAlater® Stabilization Solution has been tested on a variety of mammalian tissues, plants, E. coli, Xenopus, fish, and Drosophila. It has been successfully used with all of our Ambion® RNA isolation kits (see figure below) including RNAqueous® Total RNA Isolation Kits, the ToTALLY RNA™ Total RNA Isolation Kit, RNAwiz™ Reagent, and Poly(A)Purist™ Kits, as well as Molecular Research Center's TRI Reagent® products. Other commercial RNA isolation kits and standard RNA isolation protocols are also compatible with RNAlater® Stabilization Solution.

Trim the tissue to be less than 0.5 cm in at least one dimension and simply submerge it in 5 volumes of RNAlater® Stabilization Solution (e.g., a 0.5 g sample requires about 2.5 mL of RNAlater® Stabilization Solution). Small organs such as rat kidney, liver, and spleen can be stored whole in RNAlater® Stabilization Solution. Resuspend pelleted cells in a small amount of PBS before adding 5–10 volumes of RNAlater® Stabilization Solution. You can store samples at 4°C for one month, at 25°C for one week, or at –20°C indefinitely. You can archive tissues treated with RNAlater® Stabilization Solution at –20°C.

For RNA isolation, simply remove the tissue from RNAlater® Stabilization Solution and treat it as though it was just harvested. Most tissues can be homogenized directly in lysis buffer, although harder tissues such as bone may require freezing in liquid nitrogen and grinding.

For your tissue samples, we recommend removing the tissue treated with RNAlater® Stabilization Solution from the excess RNAlater® solution after incubation overnight at 4°C, blotting the tissue on a sterile Kimwipe® wiper, and proceeding to lysis.

For your cell samples, pellet the cells, then pour off the excess RNAlater® supernatant. If possible, aspirate the RNAlater® Stabilization Solution from the cell pellet and proceed to lysis. Cells are generally less fragile after being stored in RNAlater® Stabilization Solution and can be centrifuged at higher speeds without causing cell lysis (5,000 x g).

Yes, DNA can be isolated from samples stored in RNAlater® Stabilization Solution. Please view the protocol here. Proteins will be preserved in RNAlater® Stabilization Solution; however, the solution will denature the proteins. Therefore, the protein obtained from samples stored in it will still be suitable for applications such as western blotting or 2D gel electrophoresis, but not for applications that require native protein.

RNA was evaluated after storage for over 2 years, indicating no systematic bias introduced by storage in RNAlater® Stabilization Solution. For more information, visit our webpage.

RNAlater® Stabilization Solution is bacteriostatic. Although bacteria does not grow in RNAlater® solution, the cells remain intact. E. coli cells stored in RNAlater® Stabilization Solution for 1 month at 4°C are intact and yield undegraded RNA.

Yes, RNAlater® Stabilization Solution has been used with Staphylococcus epidermidis isolates. Here’s a reference describing how researchers used the solution with gram-positive bacteria:
Handke LD, Conlon KM, Slater SR et al. (2004) Genetic and phenotypic analysis of biofilm phenotypic variation in multiple Staphylococcus epidermidis isolates. J Med Microbiol 53(Pt5):367–344.

No, the tissue in RNAlater® Stabilization Solution must be stored at 4°C overnight.

Yes, you should be able to sort cells by fluorescence-activated cell sorting (FACS) after treatment with RNAlater® Stabilization Solution. If you run into problems due to the viscosity of RNAlater® Stabilization Solution, you may need to dilute it with cold nuclease-free water. This will not affect the protection of the RNA. Samples should be processed quickly and as cold as possible. Read more about this procedure and view references here. You should also be able to perform mass spectrophotometry on samples treated with RNAlater® Stabilization Solution. Please note that salt can form adducts on the protein at the desorption/ionization step. To avoid this problem, dialyze the sample to get rid of all salt.

RNA stored in RNAlater® Stabilization Solution is stable for 1 day at 37°C, 1 week at 25°C, 1 month at 4°C, or long-term at –20°C.

We do not recommend this, as RNAlater® Stabilization Solution is already at a 1X concentration.

RNAlater®-ICE Frozen Tissue Transition Solution can be used to submerge a frozen sample, then thaw it overnight at –20°C or colder. Once thawed, tissues can then be processed like fresh tissues using standard RNA isolation procedures.

Nucleases

Nuclease

Source

Cleavage specificity

Required cofactors

Comments

RNase A

Bovine

3’ of single-stranded C's and U's

None

Inactivated by 0.1% SDS

RNase I

E. coli (recombinant)

3’ of single-stranded NTPs of RNA

None

Responsible for 98% of the EDTA-resistant RNase activity in E. coli extracts

RNase T1

Aspergillus oryzae (recombinant)

3’ end of single-stranded G's

None

Specificity for G is 10^6 times greater than A

RNase V1

Cobra venom

dsRNA

Na+ (100 mM); Mg2+ (0.3 mM)

Cleaves ssRNA at 10% of the rate of dsRNA

RNase H

E. coli (recombinant)

RNA of RNA:DNA hybrids to give 5’-phosphate terminated oligoribotides

Mg2+ (2–4 mM)

EDTA abolishes activity, but enzyme can still bind RNA:DNA hybrids. RNase H will not degrade DNA or unhybridized RNA

Total RNA can be digested using RNAse A or RNase T1. A mix of both can be used in ribonuclease protection assays.

Magnesium and calcium are required cofactors for DNase I. DNase I does digest ssDNA. To learn more about DNase I, click here.

DNase can be removed by the following methods: acid phenol:choloform extraction, lithium chloride precipitation, EDTA, and heat inactivation, or using our MegaClear™ Transcription Clean-Up Kit (Cat. No. AM1908).

Alternatively, our Ambion® TURBO DNA-free™ Kit (Cat. No. AM1907) is supplied with a DNase inactivation reagent that can be used to remove the DNase, as well as divalent cations such as magnesium and calcium, which can catalyze RNA degradation when RNA is heated with the sample.

Yes, while a single DNase treatment is typically sufficient for a vast majority of DNA removal needs, a second treatment option is available. Please see the protocol here.

RNA Isolation - General

Yes, please visit this website for our FirstChoice® total RNA isolated from human tissues, as well as from E. coli and human cell lines.

The three salts that can be used are:

  • Guanidine thiocyanate, which requires ethanol. Guanidine thiocyanate is a common agent used for isolating RNA and we recommend it especially for tissues high in ribonuclease activity, such as the pancreas or spleen.
  • Ammonium acetate, which requires ethanol. Ammonium acetate is useful when reducing coprecipitation of unwanted dNTPs and oligosaccharides. However, it should not be used when the nucleic acid will be phosphorylated using T4 polynucleotide kinase, since this enzyme is inhibited by ammonium ions.
  • Lithium chloride, which does not require ethanol. LiCl is very effective in precipitating RNA but does not efficiently precipitate protein or DNA. It also does not precipitate unincorporated nucleotides.

RNA and DNA absorb at 260 nm while protein absorbs at 280 nm. The A260/A280 absorbance ratio can be used to indicate the purity of the sample. For RNA, the A260/A280 ratio should be approximately 2.0. If the ratio is lower, this indicates protein or phenol contamination of the sample.

The A260/A230 ratio is a measure for pure nucleic acids, with an expected range between 2.0 and 2.2. If the ratio is much lower than this, it indicates the presence of contaminants in the sample (typically phenol, guanidine, ethanol, or glycerol used for precipitation).

The 28S and 18S bands are indicative of intact RNA. On a gel, the 28S and 18S bands should be present in an approximately 2:1 ratio.

UV spectroscopy is the traditional method for assessing RNA concentration and purity. The Agilent® 2100 Bioanalyer® instrument combines the use of microfluidics, capillary electrophoresis, and a fluorescent dye that binds to nucleic acid to evaluate both RNA concentration and integrity. Read more about both methods here.

For bacterial samples, you could use our TRIzol® Max™ Bacterial RNA Isolation Kit (Cat. No. 16096040), that contains an extra reagent during homogenization to help with lysis of the bacterial cell walls. You could also use our PureLink® RNA Mini Kit (Cat. No. 12183018A), for which you would need to prepare a lyosyme solution to lyse the cell wall.

Yes, you can use TRIzol® Reagent or the mirVana™ PARIS™ RNA and Native Protein Purification Kit.

Please visit our website for tips for working with blood samples.

RNA Isolation Kits

The absorbance of nucleic acids is dependent upon the ionic strength and pH of the medium. Please see the range of absorbance values below based on the diluents used.

Diluent

A260

A280

A260/A280

RNA (µg/mL)

Cytoplasmic RNA dissolved in distilled water

0.381

0.223

1.711

15.24

Cytoplasmic RNA dissolved in TE buffer

0.335

0.145

2.310

13.4

RNA isolated by TRIzol® Reagent and dissolved in distilled water

0.585

0.328

1.785

23.4

RNA isolated by TRIzol® Reagent and dissolved in TE buffer

0.544

0.247

2.206

21.76

Although a high A260/A280 ratio may not indicate an extremely pure preparation of nucleic acid, a low A260/A280 ratio (1.7 for RNA) does indicate that the preparation is contaminated and may not be suitable for some applications.

You can homogenize your samples using a glass, Teflon®, or power homogenizer (Polytron® or Tekmar’s Tissumizer® homogenizer). You do not need to homogenize cultured cells, just mix them extensively with TRIzol® Reagent. Sonication will work to lyse cells in TRIzol® Reagent. For small samples, you can try using a Dounce homogenizer.

There are several possible stopping points and recommended storage conditions during the extraction of RNA with TRIzol® Reagent:

  • Sample homogenization step: After homogenization (before addition of chloroform), you can store samples at –70°C for at least 1 year. The homogenated samples can sit at room temperature for several hours before adding chloroform.
  • Sample homogenization step: If samples are efficiently lysed in TRIzol® Reagent and the reagent inactivates the nucleases, you can safely store RNA for 3–4 days at room temperature.
  • RNA precipitation step: You can store RNA in isopropanol overnight at 4°C. Prolonged storage at this reduced temperature will not influence the yield of RNA appreciably. Do not store at –20°C, as salts will precipitate, and do not store for a prolonged time at room temperature because the guanidine isothiocyanate can harm the RNA.
  • RNA wash step: You can store RNA in 75% ethanol for 1 week at 4°C or 1 year at –20°C.

Small volumes (0.5–0.8 mL) of TRIzol® Reagent have been used successfully for 102 to 105 cells, but if small volumes are used, we recommend using smaller tubes in order to have the tallest possible column of aqueous phase. The taller the column of liquid, the less likely that contamination from the interphase will occur.
Here is a protocol for isolation of RNA from small quantities of tissue (1–10 mg) or cells (100–10,000):

  1. Add 800 μL TRIzol® Reagent to the sample. Homogenize cells by pipetting repeatedly. Add 200 μg glycogen (Cat. No. 10814010) directly to the TRIzol® Reagent. If processing tissue, pulverize in liquid nitrogen first and then add 800 μL TRIzol® Reagent containing 200 μg glycogen (final concentration 250 μg/mL) followed by vigorous vortexing or power homogenization.
  2. Place at room temperature, cap the vial, and vortex at high speed for 10 seconds. Make sure the TRIzol® Reagent wets the side of the vial in order to solubilize any sample that may be remaining on the walls.
  3. Shear the genomic DNA in the sample by passing twice through a 26-gauge needle connected to a 1 mL syringe. Using the syringe, transfer the sample to a sterile 1.5 mL microcentrifuge tube.
  4. Add 160 μL of chloroform (or 49:1 chloroform:isoamyl alcohol) to each sample and vortex up to 30 seconds. Centrifuge at maximum speed in a microcentrifuge for 5 minutes to separate the phases.
  5. Transfer the upper aqueous phase to a fresh tube and add 400 μL ice-cold isopropanol. Allow the samples to precipitate at –20°C for 1 hour to overnight. Pellet the RNA by centrifugation at maximum speed in the microfuge for 15 minutes at room temperature.
  6. Decant the supernatant. Wash the pellet in 200 μL of 70% ethanol and centrifuge again for 10 minutes at maximum speed. Decant the supernatant, removing as much as possible without disturbing the pellet. Dry the RNA pellet.
  7. Resolubilize the pellet in 30–50 μL RNAse-free deionized water. If tissue is high in RNAses (e.g., adrenal gland, pancreas), resuspend in 100% deionized formamide. Be sure to vortex or pipette the sample up and down to ensure that the pellet is fully resolubilized. Store at –70°C.

We recommend using straight chloroform. No isoamyl alcohol is needed (though using chloroform:isoamyl alcohol 49:1 works without problems). You can also use chloroform with 50 ppm amylene. Alternatively, BCP (1-bromo-2 chloropropane) can be used in the place of chloroform.

TRIzol® LS Reagent is designed to be used with liquid samples, such as serum or virus preparations, while TRIzol® Reagent is designed to be used with tissues or cells, including lipid-rich and difficult samples.

The TRIzol® Plus RNA Purification Kit combines the lysis capability of TRIzol® Reagent with the convenient RNA extraction technology of the silica spin columns included in the PureLink® RNA Mini Kit. Learn more about the system here, or view a workflow schematic here.

The PureLink® RNA Mini Kit was only validated to recover RNA > 200 nt. However, you can try the following modifications to isolate total RNA including small RNA:

  • Add more ethanol to the lysate before loading it onto the column. The final ethanol concentration should be at least 55%.
  • Increase the ethanol concentration in Wash 1 to at least 55%.
  • No on-column DNase treatment can be used without loss of the small RNA.

For on-column digestion, PureLink® DNase (Cat. No. 12185010) can be used. Please see the protocol in the appendix of the manual on page 63.

The PureLink® RNA Mini Kit provides rapid column-based purification of total RNA, without organic lysis (phenol/chloroform). You can obtain up to 1,000 µg of purified RNA from a single extraction. The RiboPure™ RNA Purification Kits combine a phenol/guanidine thiocyanate solution with a glass-fiber filter purification method.

The column cut-off size is 200 nt. Using a RiboPure™ kit is not appropriate for miRNA recovery. For miRNA isolation, we would suggest using a mirVana™ kit, the TRIzol® Plus RNA Purification Kit, or the PureLink® RNA Micro Kit.

RNaqueous® kits are based on a rapid, filter-based RNA isolation system that does not require phenol, chloroform, or other toxic organic chemicals. The RNAqueous® Total RNA Isolation Kit is designed for sample sizes of 10–75 mg of tissue or 106 to 107 cells, whereas the RNAqueous®-Midi Total RNA Isolation Kit is designed for tissue samples of up to 0.5 g or 109 cells. The RNAqueous®-Micro Total RNA Isolation Kit is optimized for the purification of total RNA from micro-sized samples such as 10–500,000 cultured cells, as few as 10 microdissected cells, or up to 10 mg tissue. The RNAqueous®-96 Total RNA Isolation Kit utilizes a 96-well plate format for high-throughput RNA isolation from 100 to 2 x 106 cells or 0.1 to 1.5 mg of tissue. The RNAqueous®-4PCR Total RNA Isolation Kit is designed for RNA isolation from small tissue samples (0.5–75 mg) and small- to medium-sized cultured cell samples (~100–107 cells).

No, please follow the RNA isolation methods described with the Tempus® system. The collection tubes are not compatible with the RiboPure™ kit.

The former (Cat. No. AM1928) is for isolation of RNA from human blood and the latter (Cat. No. AM1951) is for isolation of RNA from mouse and rat blood. Both kits have the same columns but differ in protocol and the wash 1 buffer, wash 2/3 buffer, and sodium acetate solutions. We do not recommend using the mouse kit for human blood samples. The mouse kit accommodates the difference in viscosity of mouse/rat vs. human blood and also the higher mouse and rat blood RNA yield. The mouse kit allows recovery of either total RNA containing small RNAs or total RNA depleted of small RNAs based on which protocol in the manual was followed. With the RiboPure™ Blood Purification Kit, using the standard protocol in the manual, you can isolate total RNA depleted of small RNAs, but with an alternate protocol, you can isolate total RNA containing small RNAs. Alternatively, if you need small RNAs alone, then you should follow the above alternate protocol for isolation of total RNAs containing small RNAs, and then follow Section IVA in the mirVana miRNA Isolation Kit manual (Cat. No AM1560).

RNA recovered using the LeukoLOCK™ Total RNA Isolation System typically contains less than 10% of the amount of reticulocyte-derived alpha- and beta-globin mRNAs present in typical mRNA samples from unfractionated whole blood. The isolated RNA is specially suited for gene expression profiling, as it contains a lower proportion of globin mRNA. We sell LeukoLOCK™ Fractionation and Stabilization Kit (Cat. No. AM1933) for blood collection and stabilization. You can then use LeukoLOCK™ Total RNA Isolation Kit (Cat. No. AM1934) to isolate RNA stabilized on the LeukoLOCK™ filters. Cat. Nos. AM1934 and AM1933 are available together, packaged in a single kit as the LeukoLOCK™ Total RNA Isolation System (Cat. No. AM1923).

RNA yield is typically 10–20 µg from 10 mL of blood. RNA quality depends on the quality of the blood. Typically, the RNA integrity number (RIN) value is around 9, and the 28S/18S ratio is around 1.5.

No. The kit has not been validated for less than 3 mL of human blood. It is best to use 9–10 mL blood.

The LeukoLOCK™ Total RNA Isolation System has been tested only for human blood.

We have processed blood stored with heparin with the LeukoLOCK™ Total RNA Isolation System. The RNA looks fine. But we have not tested it for any downstream applications. We discourage your use of heparin, as heparin notoriously inhibits downstream applications such as RT-PCR.

Here is a list of DNA/RNA Purification replacement buffers we offer as standalone.

Cells-to-CT™ kits

Yes, we offer our Cells-to-CT™ kits that allow you to bypass RNA purification. Read more here.

Please visit this webpage to see the cell lines we have tested. There is no reason why the Cells-to-CT™ system shouldn’t work with any cell line. However, due to differences in cell size and composition, the maximum number of cells per lysis reaction may be slightly different for different cell lines. You can test for inhibition and minimal sample input using the TaqMan® Cells-to-CT™ Control Kit.

We have tested the reagents that are stored frozen with five to ten freeze-thaw cycles and have seen no effect on CT values. Up to five freeze-thaw cycles for the lysate samples have not had any significant effect on gene expression data.

We do not recommend normalizing to an endogenous control, due to the biological variation and transcriptional noise exhibited by single cells. Because of this variation, normalization can actually increase the spread of calculated expression levels in single cells.

You can place samples at room temperature for up to 30 minutes following lysis, and up to 2 hours after adding stop solution. You can freeze cell lysates, cDNA, and preamplification samples for future processing.

We have not used it on plant tissue internally. When working with plants, we suggest one of the following standard homogenization methods: (1) a motorized homogenizer, (2) stainless steel bead beating, or (3) grinding tissues in liquid nitrogen using a mortar and pestle.

Yes. Cells-to-CT™ kits are compatible with multiplex qRT-PCR assays. If a large number of targets are being tested, we recommend combining them with TaqMan® Assays.

Cells-to-CT™ technology features a unique method for lysing cultured cells while removing genomic DNA and preserving RNA integrity. Therefore, if you follow the lysis/DNase treatment steps in the protocol, genomic DNA contamination will not be an issue for your qRT-PCR.

Yes, Cells-to-CT™ kits can be used with TaqMan® Low Density Arrays.

Yes, as shown in the graph below, both the Power SYBR® Green Cells-to-CT™ Kit and the Fast SYBR® Green Cells-to-CT™ Kit were compared to a traditional RNA purification method followed by real-time PCR analysis. Both kits show equivalent performance to results obtained using purified RNA over a broad set of gene targets. Read more about the SYBR® Green Cells-to-CT™ kits here.

No. However, the SYBR® Cells-to-CT™ reverse transcriptase will work with the TaqMan® mastermixes (but not the other way around).

Here are the potential stopping points:

• At the Cell Lysis step after addition of the Stop Solution and incubating for 2 minutes at room temperature. Do not allow lysates to remain at room temp. for longer than 20 minutes after adding the Stop Solution. Lysates can be stored on ice for a maximum of 2 hours, or at -20 degrees C or -80 degrees C for a maximum of 5 months. 

• At the Reverse Transcription (RT) step after assembling the RT reaction. Once assembled, mix reactions gently, then centrifuge briefly to collect the contents at the bottom of the reaction vessel, and store at 4 degrees C for up to 4 hours.

• Completed RT reactions may be stored at -20 degrees C.

The TaqMan® Gene Expression Cells-to-CT™ Kit can accommodate 45% of the total reverse transcriptase reaction volume as cell lysate.