RNSol H Reagent

RNSol H Reagent is used to purify RNA and miRNA from animal tissue and cell, PBMC, whole blood, WBC and bacteria. The assay is intended for in vitro diagnostic use. We recommend all users to study RNA experiments guideline, before starting their work.

The RNSol H reagent is designed for isolating all form of RNA molecules from 0.5- 100 mg tissue samples or up to 1×107 animal cells and up to 2 x 109 bacteria. Fresh or frozen tissue samples should be lysed and homogenized. Aqueous phase is separated using chloroform. Then, RNA selectively will be isolated by the addition of chloroform. Contaminants removed by 75% ethanol. Pure RNA is finally eluted in nuclease free water. Isolated RNA is ready to use in downstream applications. It has A260/A280 ratios of 1.9–2.3 by spectrophotometer, confirming high purity. 

Recommended Starting Material

To reach optimized results it is better to follow as listed below. The size of the recommended starting material to use with determined RNSol H Reagent and chloroform volumes, are written in the bellow Table.

Table Appropriate sample size and amount of RNSol H Reagent, chloroform and ethanol amount

Sample Preparation – Animal Tissue

  Storage: Fresh or frozen tissues may be used for the procedure. For long storage of tissue samples, Tissues should be flash-frozen in liquid nitrogen and transferred immediately to a -70 °C freezer. Tissues may be stored at -70 °C for several months. When isolating RNA and miRNA from frozen tissues ensure that the tissue does not thaw during weighing or prior to RNA preparation. It is recommended to store tissue sample in RNaseLag for storage (refer to RNaseLag).

   Preparation: Sample preparation is composed of 2 steps, disruption and homogenization. First step is disruption of cell walls, plasma membranes of cells and organelles. Inefficient disruption results in RNA yield reduction. This can be done with one or cooperation of two methods depending on the tissue type. These methods include RNSol H Reagent, Mortar and Pestle, Tissuelyser etc. Viscosity reduction can be accomplished by Homogenization. The aim is to create a homogeneous lysate. Sometimes these 2 steps happen simultaneously. For more information refer to the bellow Table.

Note: After disruption and homogenization in RNSol H Reagent (lysis buffer), samples can be stored at –70°C for months.

Table . Disruption and homogenization for different sample types

Optional: Homogenizing step is optional. It means that using homogenizer or syringe needle to homogenize the lysate can be omitted from the process.

RNaseLag

 For optimum result, it is recommended to store tissue samples in RNaseLag. RNaseLag is a Reagent which stabilizes RNA in tissues and cells.

Procedure

• Cut the animal tissue sample into slices less than 5mm thick, as quickly as possible.
• Completely immerse the tissue pieces in the collection vessel containing RNaseLag.

Note: Make sure to use the appropriate volume of RNaseLag, so weight your sample before starting the procedure and use 10µl RNaseLag per 1mg of tissue.

• The sample is ready for archival storage at conditions shown in the bellow Table .
• After storage, for RNA isolation continue with appropriate protocol for the chosen sample type.

Table Storage conditions and procedures after RNaseLag treatment. 

Sample Preparation-Animal Cell

Storage: Fresh or frozen samples may be used by RNall. Frozen samples can be kept at -80°C for long time. As a guide, storage preparation stock and conditions are written here.

Cell selection: First, ensure that the cells are in their best possible condition. Select cultures near the end of log phase growth (approximately %90 confluent) and change their medium 24 hours prior to harvesting. Carefully examine the culture for signs of microbial contamination. Facilitate this by growing cultures in antibiotic-free medium for several passages prior to testing. This allows time for any hidden, resistant contaminants (present in very low numbers) to reach a higher, more easily detected level. Samples of these cultures are then examined microscopically and tested by direct culture for the presence of bacteria, yeasts, fungi, and mycoplasmas.

Cell harvesting:  Remove all dissociating agents by washing or inactivation (especially important when using serum-free medium). Centrifugation, when absolutely necessary, should only be hard enough to obtain a soft pellet; 100 x g for 5 to 6 minutes is usually sufficient. Count and then dilute or concentrate the harvested cell suspension to twice the desired final concentration, which is usually 4 to 10 million viable cells per milliliter. An equal volume of medium containing the cryoprotective agent at twice its final concentration will be added later to achieve the desired inoculum. Keep the cells chilled to slow their metabolism and prevent cell clumping. Avoid alkaline pH shifts by gassing with CO2 when necessary.

Cryoprotection: Cryoprotective agents are necessary to minimize or prevent the damage associated with slow freezing. DMSO is most often used at a final concentration of 5 to %15 (v/v). Always use reagent or other high purity grades that have been tested for suitability. Sterilize by filtration through a 0.2-micron nylon membrane in a polypropylene or stainless-steel housing and store in small quantities (5ml). Some cell lines are adversely affected by prolonged contact with DMSO. This can be reduced or eliminated by adding the DMSO to the cell suspension at 4°C and removing it immediately upon thawing. If this does not help, lower the concentration or try glycerol or another cryoprotectant. Glycerol is generally used at a final concentration of between 5 and %20 (v/v). Sterilize by autoclaving for 15 minutes in small volumes (5ml) and refrigerate in the dark. Although less toxic to cells than DMSO, glycerol frequently causes osmotic problems, especially after thawing. Always add it at room temperature or above and remove slowly by dilution. High serum concentrations may also help cells survive freezing. Replacing standard media-cryoprotectant mixtures with %95 serum and %5 DMSO may be superior for some overly sensitive cell lines, especially hybridomas. Add cryoprotective agents to culture medium (without cells) immediately prior to use to obtain twice the desired final concentration (2X). Mix this 2X solution with an equal volume of the harvested cell suspension to obtain the inoculum for freezing. This method is less stressful for cells, especially when using DMSO as the cryoprotectant.

Cooling rate: The cooling rate used to freeze cultures must be just slow enough to allow the cells time to dehydrate, but fast enough to prevent excessive dehydration damage. A cooling rate of -1°C to -3°C per minute is satisfactory for most animal cell cultures. Larger cells, or cells having less permeable membranes may require a slower freezing rate since their dehydration will take longer. Transfer from the cooling chamber or device to the final storage location must be done quickly to avoid warming of the vials. Use an insulated container filled with dry ice or liquid nitrogen as a transfer vessel to ensure that the cells remain below -70°C.

Thawing: Remove the vial from its storage location and carefully place the vessel in warm water, agitate gently until completely thawed. Rapid thawing (60 to 90 seconds at 37°C) provides the best recovery for most cell cultures; it reduces or prevents the formation of damaging ice crystals within cells during rehydration.

Recovery: Since some cryoprotective agents may damage cells upon prolonged exposure, remove the agents as quickly and gently as possible. Several approaches are used depending on both the cryoprotective agents and characteristics of the cells. Most cells recover normally if they have the cryoprotective agent removed by a medium change within 6 to 8 hours of thawing. Transfer the contents of the vial to a T-75 flask or other suitable vessel containing 15 to 20 milliliters of culture medium and incubate normally. As soon as a majority of the cells have attached, remove the medium containing the now diluted cryoprotective agent and replace with fresh medium.

For cells that are sensitive to cryoprotective agents, removing the old medium is easily accomplished by gentle centrifugation. Transfer the contents of the vial to a 15ml centrifuge tube containing 10ml of fresh medium and spin for 5 minutes at 100 x g. Discard the supernatant containing the cryoprotectant and resuspend the cell pellet in fresh medium. Then transfer the cell suspension to a suitable culture vessel and incubate normally.

When glycerol is used as the cryoprotectant, the sudden addition of a large volume of fresh medium to the thawed cell suspension can cause osmotic shock, damaging or destroying the cells. Use several stepwise dilutions with an equal volume of warm medium every 10 minutes before further processing to give the cells time to readjust their osmotic equilibrium.

Preparation: It is crucial to use the correct amount of starting material. RNA content can vary greatly from cell to cell. Therefore, counting cells is the most important step before starting the procedure (for more information refer to appendix 7). However, as a guide, the number of HeLa cells after confluent growth obtained in various culture vessels, is given in the bellow Table . After counting and selecting the intended cell volume, refer to the previous Table for disruption and homogenization guide of cells.

Table Number of HeLa cells in various culture vessels

Maximize RNA Yield

To obtain higher yield of RNA, it is important to follow protocols carefully and pay attention to sample size table and its suitable lysis buffer amount recommended for your samples. Notice that all samples must be completely homogenized during cell lysis for maximum yields.  It is good to know that:

• Yield and quality of the purified RNA depends on sample storage conditions. For best results, it is recommended to use fresh samples, however for long storage, it is better to treat sample in RNaseLag (for more information refer to RNaseLag).
• Avoid freezing and thawing samples, which may result in decreasing RNA yield, as compared to isolating RNA from fresh samples.

Sample preparation–Bacteria

Typical yields of RNA will vary depending on the cell density of the bacterial culture and the bacterial species, hence before starting, it’s recommended to determine your bacterial species. As a guide, bacteria culture preparation and storage conditions are written here.

Storage

Fresh or frozen bacteria samples may be used by RNall. Frozen samples can be kept at -80°C for a long time. As a guide, storage, preparation of stock and conditions are written here.

Bacteria culture

The following protocol is for inoculating an overnight culture of liquid LB with bacteria.

• Prepare liquid Luria-Bertani (LB)

To make 400 mL of LB, weigh out the following into a 500 mL glass bottle:

• 4 g NaCl

• 4 g Tryptone

 • 2 g Yeast Extract

• and dH2O to 400 mL

Loosely close the cap on the bottle and then loosely cover the entire top of the bottle with aluminum foil. Autoclave and allow to cool to room temperature. Now screw on the top of the bottle and store the LB at room temperature.

• Use a single, well-isolated colony from a fresh Luria-Bertani (LB) agar plate to inoculate 1–10 ml of LB medium.
• Loosely cover the culture with sterile aluminum foil or a cap that is not airtight.
• Incubate bacterial culture at 37°C for 12-18 hour in a shaking incubator

Storing condition

• Autoclave microcentrifuge tube or 1-3 ml screw cap.
• Grow a fresh overnight culture of the strain in broth. Do not grow the cultures too long. Bacteria strains should be grown to late log phase.
• Label the tube with the strain and date.
• Either 5% to 10% DMSO or glycerol can be used as cryopreservation in the culture medium. Glycerol is usually prepared in aqueous solution at double the desired final concentration for freezing. It is then mixed with an equal amount of cell suspension.
• Aliquot 1 to 1.8 ml of bacteria to each vial and seal tightly with screw cap.
• Allow the cells to equilibrate in the freeze medium at room temperature for a minimum of 15 min but no longer than 40 min. After 40 min, the viability may decline if DMSO is used as the cryoprotectant.
• Place the vials into a pre-cooled (4°C), controlled rate freeze chamber and place the chamber in a mechanical freezer at -70 °C for at least 24 hours.
• Quickly transfer the vials to liquid nitrogen or at -130 °C freezer. After 24 hours at -130 °C, remove one vial, restore the bacteria in the culture medium and check viability and sterility.

Recovery of cryopreserved cells

• Prepare a cultured vessel that contains at least 10 ml of the appropriate growth medium equilibrate for both temperature and pH.
• Remove the vial containing the strain of interest and thaw by gentle agitation in a 37 °C water bath (or a bath set at the normal growth temperature for that bacterial strain). Thaw the strain rapidly until all ice crystals have been melted (approximately 2 min).
• Remove the vial from the bath and decontaminate it by dipping in or spraying with 70% ethanol. Unscrew the top of the vial and transfer the entire content to the prepared growth medium. Examine the cultures after an appropriate length of time. If the broth shows growth in 1-2 days, streak a plate from the broth and verify that is the correct strain.

Preparation: It is crucial to use the correct amount of starting material. RNA content can vary greatly between different bacteria types. So, counting cells is the most important step before starting the procedure. The input bacterial cell amount should not exceed 2 × 10^9 cells. For example, for E. coli, depending on culture growth, this is equivalent to 0.5 – 1.0 mL of an overnight culture. It is not recommended to exceed 1 mL of culture for this procedure. It is important to measure bacterial growth by spectrophotometer before starting the protocol. (For cell counting guideline refer to appendix 7). After counting and selecting the intended cell volume, refer to Table 3 for disruption and homogenization guide.

RNaseLag

For optimum results, it is recommended to store your samples in RNaseLag. RNaseLag is a Reagent, which provides in vivo stabilization of RNA in bacteria to ensure reliable gene expression analysis.

Procedure

• Calculate the required volume of bacterial culture.
• Add two volumes of RNaseLag into a tube.
• Add one volume of bacterial culture to the tube. Mix by vortexing for 5 sec. Incubate at room temperature (15–25°C) for 5 min.
• Centrifuge for 10 min at 4000 rpm at universal centrifuge.

Note: Sometimes the pellet is too clear to be recognized, it is due to RNaseLag treatment, and will not affect the ongoing process.

• Decant the supernatant.

Pellets can be stored at –20 to –30 °C for up to 2 weeks or at –70 °C for up to one month. For RNA isolation, thaw pellets at room temperature (15–25 °C) and proceed the appropriate RNA isolation protocol.

Genomic DNA Contamination

RNall is designed to selectively isolate the RNA. However, if further DNA removal is intended, it is recommended to use DNase Treatment kits, which are available from different suppliers.

Before start

Not forget to add the appropriate amount of ethanol (molecular biology grade, 96–100%) to TWB1 and TWB2 as indicated on the bottle, before using for the first time. Refer to washing buffer preparation section.

Maximize RNA Yield

To obtain higher yield of RNA, it is important to follow protocols carefully and pay attention to sample size table and its suitable lysis buffer amount recommended for your samples. Notice that all samples must be completely homogenized during cell lysis for maximum yields. It is good to know that:

• Yield and quality of the purified RNA depends on sample storage conditions. For best results, it is recommended to use fresh samples, however for long storage, it is better to treat sample in RNaseLag (for more information refer to RNaseLag).

Avoid freezing and thawing samples, which may result in decreasing RNA yield, as compared to isolating RNA from fresh samples.

RNSol H Reagent is tested against predetermined experiments on a lot-to-lot basis according to ISO-certified quality management system, to ensure consistent product quality. For your information, the results of all experiments are accessible by addressing REF and Lot number on web at http://amorph.tech.

Shipment condition is checked by Carbon Technologies. After arrival, RNSol H Reagent should be kept dry, at 2-8 °C. When storage condition is as directed, all reagents are stable until expiration date, as indicated on the kit box.

Due to chemical material usage that may be hazardous, always make sure to wear suitable lab coat, disposable gloves, and protective eyewear. Material Safety Data Sheet (MSDS) for all products and reagents are provided. They are accessible online at https://amorph.tech.