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测序经验谈之:原版RNAlater Solution使用说明书  

2014-09-13 00:11:44|  分类: 二代测序 |  标签: |举报 |字号 订阅

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RNAlater Solution - 陈云地 - 现代汉语的音韵美感寻找
 

 

RNAlater Tissue Collection: RNA Stabilization Solution

 

■ Product Description
■ Product Information
■ Guidelines for Use of RNAlater Solution
■ Storage in RNAlater Solution
■ RNA Isolation from Samples in RNAlater Solution
■ Quality Control
■ Appendix A Safety Information
■ Documentation and Support

Product Description

RNAlater Tissue Collection: RNA Stabilization Solution is an aqueous tissue storage reagent that rapidly permeates most tissues to stabilize and protect RNA in fresh specimens. It eliminates the need to immediately process or freeze samples; the specimen can simply be submerged in RNAlater Solution and stored for analysis at a later date. Samples in RNAlater Solution can be stored for extended periods under conditions where RNA degradation would normally take place rapidly .Tissues can be stored indefinitely in RNAlater Solution at –20°C or below.

 

Product Information - Storage and Stability

Store RNAlater Solution at room temperature.

If any precipitation of RNAlater Solution is seen, heat it to 37°C and agitate to redissolve it.

 

Sample Types Compatible with RNAlater Solution

RNAlater Solution can be used for RNA preservation with most tissues, cultured cells, bacteria, and yeast. It may not be effective in tissues that are poorly penetrated by the solution, such as waxy plant tissue and bone. RNAlater Solution has been extensively tested with animal tissues, including brain, heart, kidney, spleen, liver, testis, skeletal muscle, fat, lung, and thymus. It has also been proven effective for RNA preservation in E. coli, Drosophila, tissue culture cells, white blood cells, and some plant tissues. For more information, go to

www.invitrogen.com/site/us/en/home/support/technical-support.html.

 

RNA Isolation from RNAlater Solution

RNAlater Solution is compatible with most RNA isolation methods. Samples stored in RNAlater Solution have been used successfully with TRI Reagent Solution (P/N AM9738), and all of Ambion RNA isolation kits and reagents, including: the TōTALLY RNA Kit, the PARIS? Kit, the mirVana miRNA Isolation Kit, and the

RNAqueous and Poly(A)Purist product families.

 

Isolating Genomic DNA from RNAlater Solution-stored Samples

DNA can be isolated from RNAlater Solution-stored samples. For more information, go to www.invitrogen.com/site/us/en/home/support/technical-support.html.

 

Isolating Protein from RNAlater Solution-stored Samples

Proteins are also preserved in RNAlater Solution. RNAlater Solution will denature proteins; therefore, protein obtained from samples stored in it will be suitable for applications such as Western blotting or 2D gel electrophoresis, but not for applications that require native protein.

 

Guidelines for Use of RNAlater Solution

 

· Use RNAlater Solution with fresh tissue only; do not freeze tissues before immersion in RNAlater Solution.

· Before immersion in RNAlater Solution, cut large tissue samples to ≤ 0.5 cm in any single dimension.

· Place the fresh tissue in 5-10 volumes of RNAlater Solution.

· Most samples in RNAlater Solution can be stored at room temperature for 1 week without compromising RNA quality, or at –20°C or –80°C indefinitely.

· Do not freeze samples in RNAlater Solution immediately; store at 4°C overnight (to allow the solution to thoroughly penetrate the tissue), remove supernatant, then move to –20°C or –80°C for long-term storage.

Note: We offer RNAlater-ICE (P/N AM7030) to recover tissues that have already been frozen. RNAlater-ICE renders frozen tissues pliant enough for homogenization while maintaining the low temperatures needed to protect the RNA from degradation.

 

Animal Tissue

RNAlater Solution does not disrupt the structure of tissues; thus, tissue that has been equilibrated in RNAlater Solution can be removed from the solution, sectioned into smaller pieces, and returned to RNAlater Solution, if desired. Small organs such as mouse liver, kidney and spleen can be stored whole in RNAlater Solution.

 

Plant Tissue

Plant tissues that have natural barriers to diffusion, such as waxy coatings on leaves, will often require disruption to allow RNAlater Solution access to the tissue. However, many plant tissues can simply be submerged in RNAlater Solution whole; we have successfully isolated intact RNA from tobacco leaf explants, entire Arabidopsis and alfalfa seedlings, and from potato shoot tips.

 

Tissue Culture Cells

Pellet cells according to the protocols followed by your laboratory. Remove supernatant and then add 5-10 volumes RNAlater Solution. The cells can be washed in PBS before resuspending in RNAlater Solution, if desired.

 

Blood and Plasma

White blood cells can be effectively preserved in RNAlater Solution when separated from the red blood cells and sera and treated as tissue culture cells. RNAlater Solution can also be added to small volumes of anticoagulated whole blood, sera, and plasma; however, the procedure is not presented here-see the Ambion RiboPure-Blood Kit (P/N AM1928) protocol for detailed instructions.

 

Yeast

Pellet up to 3 x 108 cells (centrifuge at 12,000 x g for 2 min). Remove supernatant and immediately resuspend the pellet in 0.5-1 mL of RNAlater Solution. Yeast cells can be stored in RNAlater Solution for up to 8 hr at 25°C, or up to a week at 4°C. For long-term storage, incubate the cells in RNAlater Solution for 1 hr. Repellet the cells (centrifuge at >12,000 x g for 5 min), remove supernatant, flash freeze, and store at –80°C.

 

Bacteria

RNAlater Solution is bacteriostatic; although bacteria do not grow in it, the cells remain intact. E. coli stored in RNAlater Solution for 1 month at 4°C are intact and yield undegraded RNA.

 

Storage in RNAlater Solution

 

If refrigeration is available:

Storage at –80°C

Storage at –80°C is recommended for archival samples and will provide optimal preservation. Samples can be stored at –80°C indefinitely. RNAlater Solution will freeze at –80°C.

To prepare samples for storage at –80°C, first incubate the samples in RNAlater Solution overnight at 4°C to allow thorough penetration of the tissue, then transfer to –80°C. To expedite thawing of the samples, we recommend removing the tissue, or pelleting cells, from the RNAlater Solution before freezing at –80°C.

Samples can subsequently be thawed at room temperature and refrozen without significantly affecting the amount or the integrity of the recoverable RNA.

Storage at –20°C

Storage at –20°C can also be used for archival samples. Samples will not freeze at –20°C, but crystals may form; this will not affect subsequent RNA isolation. Samples can be stored at –20°C indefinitely.

To prepare samples for storage at –20°C, first incubate the samples in RNAlater Solution overnight at 4°C to allow thorough penetration of the tissue, then transfer to –20°C.

Samples can subsequently be thawed at room temperature and refrozen without affecting the amount or the integrity of the recoverable RNA.

Storage at 4°C

Most samples can be stored in RNAlater Solution at 4°C for up to 1 month without significant RNA degradation.

 

If refrigeration is not available:

Place samples in the coolest environment available. If ambient temperature is above 25°C, incubate the samples in RNAlater Solution on ice for a few hours, if possible, before storing at ambient temperature.

Storage at 25°C (room temperature)

Most samples can be stored at 25°C in RNAlater Solution for up to 1 week without significant loss of RNA quality. After 2 weeks at 25°C, RNA generally appears slightly degraded (marginally acceptable for Northern analysis, but still of sufficient quality for nuclease protection assays or RT-PCR analysis).

Storage at 37°C

RNA isolated from samples stored at 37°C is intact after a 24 hour incubation, but is partially degraded after 3 days.

 

RNA Isolation from Samples in RNAlater Solution

 

Remove RNAlater Solution from Samples

RNase inactivation is reversible; do not rinse RNAlater? Solution from samples before using. Blot tissues with a wipe, or pellet cells to remove excess RNAlater Solution.

Tissue

Retrieve tissue from RNAlater Solution with sterile forceps, quickly blot away excess RNAlater Solution with an absorbent lab wipe or paper towel, and then submerge the sample in RNA isolation lysis solution. Homogenize tissue promptly after placing it in lysis/denaturation solution.

Cells

There are two options for isolating RNA from cells stored in RNAlater? Solution. The preferred method is to remove the solution from the cells prior to extraction. Alternatively, cells in RNAlater? Solution can be used directly for RNA extraction. Because of the greater volume that the cells are in, this method generally requires additional lysis solution.

Removal of RNAlater Solution prior to extraction

Because of the density of RNAlater Solution, greater centrifugal forces are required to pellet cells from RNAlater Solution than from normal media. Generally, cells become much less fragile when stored in RNAlater Solution and can be centrifuged at high speed without lysis. Most cell types can be centrifuged at 5000 x g without damage to the cells. Since different cell types vary in their ability to withstand centrifugal forces, we recommend testing the centrifugal speed with an expendable sample. Alternatively, dilute the RNAlater Solution by adding an equal volume of ice cold PBS (or other buffered solution) immediately before centrifugation to reduce the density of the solution, then centrifuge at normal speeds.

RNA extraction from cells in RNAlater Solution

One-step phenol-based disruption/extraction solutions, such as Ambion TRI Reagent Solution or RNAwiz Reagent (available only in Japan), can be used to purify RNA from cells suspended in RNAlater Solution. This can be done by adding ten volumes of the one-step solution to the cell mixture, and proceeding normally. When RNAwiz Reagent is used in this way, it may be necessary to dilute the aqueous phase before the RNA precipitation step. See below for more information.

 

Tips for RNA isolation


Glass fiber-based extraction

Lysates from RNAlater Solution-treated samples often require more force to pass through glass-fiber filters than lysates from untreated samples. Therefore, it may be necessary to use centrifugation instead of vacuum pressure to pass lysates through glass-fiber filters.


One-step disruption/extraction solutions

When using one-step RNA isolation products such as TRI Reagent Solution or RNAWIZ Reagent (available only in Japan), on RNAlater Solution-preserved samples, the aqueous phase will occasionally appear cloudy; this will not adversely affect RNA recovery or quality.

With RNAWIZ Reagent, there may be a problem getting the aqueous phase to mix with isopropanol at the precipitation step because of RNAlater Solution carryover. If this occurs, add a mixture of 50% water, 50% isopropanol until the solution becomes clear and the two phases mix. The amount of water/isopropanol required will depend on how much RNAlater Solution was carried over; if the sample was mostly RNAlater Solution, as much as an equal volume may be needed.


Quality Control

RNAlater Solution undergoes quality assurance testing to verify that its composition is invariant from lot to lot.

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