Lab Work

RNA Isolation

There are a few different approaches to isolating RNA in the Roberts Lab.

Trizol/Tri-Reagent/RNA-zol: This is a classic, phenol/chaotropic salt/chloroform method. It’s the most labor intensive, error prone and potentially harmful of all the methods. Although RNA-zol eliminates the need for chloroform, it’s still based on a phenol/chaotropic salt solution. This method also has a high propensity for chemical carryover and/or low RNA yields. This is the most affordable method. These solutions are stored at 4C in FTR 209.
Spin Column Kits: There are a variety of kits which rely on charged resin columns to bind RNA. Almost all kits follow the same rough protocol: homogenize tissue in provided buffer, transfer homogenate to column, spin, wash column with some buffers, elute clean RNA into new tube. Spin column kits are the fastest, cleanest approach for RNA. They are the most expensive method. Kits are stored on the shelf near the door of FTR 213.
Hybrid: ZymoResearch makes a Direct-zol RNA isolation kit. Samples are homogenized in Trizol/Tri-Reagent/RNA-zol and then transferred to a spin column for washes/elution. Slightly cheaper than a dedicated spin column kit, but still requires usage/exposure to phenol/chaotropic salt solution. Kits are stored on the shelf near the door of FTR 213.

Homogenization

Below, you will find rough guidelines for tissue homogenization via various methodologies. Please review them to decide which is best for your situation. The examples below will need to be adapted to your experiment and your RNA isolation methodology. For example, you may be working with samples stored in a preservative solution (e.g. RNA Later or DNA/RNA Shield). The guidelines below do not address how to process these types of samples. Refer to the manufacturer’s protocols for how to process samples for your RNA isolation procedure and tissue preservation method(s).

Liquid Nitrogen (LN₂) with Mortar & Pestle

Pros

Best method for complete tissue pulverization.
Best method for sample preservation (sample never thaws).
Can be easily adapted for most tissue amounts.

Cons

Very labor intensive.
Very time consuming.
Requires LN₂ which is not readily available in our lab.

Example Procedure

Note

Prior to beginning, you’ll need to get LN₂ from UW Chemistry Stores in Bagley Hall.

Pour liquid nitrogen into a small(ish) polystyrene container (or, an ice bucket works).
Use 50mL conical to transfer LN2 to a clean mortar (this tube can just be stored in the LN2 for the duration of this entire process). This pre-chills the mortar and is important!
While the mortar is pre-chilling, put bottom portion of pestle into LN2 in the polystyrene container to pre-chill. Not too deep, though, or else the pestle handle will get too cold to hold onto.
While the mortar and pestle are pre-chilling, place rounded end of steel spatula into LN2 in the polystyrene container to pre-chill.
Transfer tissue to pre-chilled mortar.
Quickly pulverize tissue using pre-chilled pestle.
Transfer powdered tissue, using pre-chilled spatula, to storage tube (preferably pre-chilled in LN₂) and/or subset to pre-chilled, zeroed-out tube on scale for weighing.
Proceed to next sample with different clean mortar/pestle/spatula.
After all mortar/pestles/spatulas have been used, wash with soap and water (use a sponge to wash).
Submerge all utensils in a 10% bleach solution for at least 10mins.
Rinse all utensils thoroughly with DI water. Dry them thoroughly with paper towels before proceeding. Residual water will be annoying to deal with and could inhibit dowstream processes.

Tip

For more thorough (and faster) drying, use a squirt bottle filled with methanol to rinse all utensils. Do this over a LABELLED plastic “shoebox” container to collect the rinse. Set utensils aside on paper towels for a few minutes.

IMPORTANT: This should ONLY be done in the fume hood with proper PPE (e.g. gloves). Be sure to double check that no ignition sources are present! Methanol is extremely flammable. Allow collected methanol rinse to evaporate in fume hood.

Disposable Plastic Mortar & Pestle

Pros

Supplies are always on hand.
Good for small tissue amounts.

Cons

Somewhat labor intensive.
Somewhat time consuming.
Tissue might thaw during process.
Sometimes requires dry ice.
Only good for small tissue amounts.

Example Procedure

Note

If processing more than 2-3 samples, get dry ice from Biochemistry Stores (Health Sciences, J-wing basement) to prevent samples from thawing while waiting to be homogenized.

Fill disposable mortar tubes with homogenization solution (e.g. Trizol, Tri-reagent, kit homogenization buffer, etc.).

Important

Do not fill mortar tubes with more than 500uL of solution! Otherwise, displacement from the pestle will cause the tube to overflow.

Store frozen samples on ice - preferably dry ice if more than 2-3 samples.
Using small dissection scissors and/or forceps and/or small steel spatula, chip out a piece of frozen tissue.
Weigh tissue with weight boat/paper.
Transfer tissue piece to pre-filled mortar tube.
Homogenize tissue with disposable pestle. Move the pestle up and down, with twisting motions to homogenize tissue. Do this until no visible chunks of tissue remain.

Tip

If processing many samples, using a motorized Pestle Pellet will significantly ease strain on your hand/fingers and make the process faster.

Use caution to avoid splashing!!

Bullet Blender (i.e. bead beater)

Pros

High throughput (i.e. can process many samples simultaneously)
Least lab intensive of all methods.
Can handle almost all tissues sizes - very small to relatively large.

Cons

Tissue will thaw during process.
Requires dry ice.
Very loud.

Example Procedure

Review the Bullet Blender protocol in the Roberts Lab Handbook before starting.

Note

The Bullet Blender will require dry ice to keep it cool during your run(s). Get dry ice from Biochemistry Stores (Health Sciences, J-wing basement).

Pre-fill tubes with desired beads and homogenization buffer.

Tip

Prepare tubes a day or two ahead of time to save you time on the actual day you perform the extractions.

Store frozen samples on ice - preferably dry ice if more than 2-3 samples.
Using small dissection scissors and/or forceps and/or small steel spatula, chip out a piece of frozen tissue.
Weigh tissue with weight boat/paper.
Transfer tissue piece to pre-filled tube.
Pre-chill Bullet Blender.
Run Bullet Blender with desired time/intensity.

--- title: "Lab Work" format: html: code-fold: false code-tools: true code-copy: true highlight-style: github code-overflow: wrap --- # RNA Isolation There are a few different approaches to isolating RNA in the Roberts Lab. - Trizol/Tri-Reagent/RNA-zol: This is a classic, phenol/chaotropic salt/chloroform method. It's the most labor intensive, error prone and potentially harmful of all the methods. Although RNA-zol eliminates the need for chloroform, it's still based on a phenol/chaotropic salt solution. This method also has a high propensity for chemical carryover and/or low RNA yields. This is the most affordable method. These solutions are stored at 4C in FTR 209. - Spin Column Kits: There are a variety of kits which rely on charged resin columns to bind RNA. Almost all kits follow the same rough protocol: homogenize tissue in provided buffer, transfer homogenate to column, spin, wash column with some buffers, elute clean RNA into new tube. Spin column kits are the fastest, cleanest approach for RNA. They are the most expensive method. Kits are stored on the shelf near the door of FTR 213. - Hybrid: ZymoResearch makes a Direct-zol RNA isolation kit. Samples are homogenized in Trizol/Tri-Reagent/RNA-zol and then transferred to a spin column for washes/elution. Slightly cheaper than a dedicated spin column kit, but still requires usage/exposure to phenol/chaotropic salt solution. Kits are stored on the shelf near the door of FTR 213. ## Homogenization Below, you will find rough guidelines for tissue homogenization via various methodologies. Please review them to decide which is best for your situation. The examples below will need to be adapted to your experiment and your RNA isolation methodology. For example, you may be working with samples stored in a preservative solution (e.g. RNA Later or DNA/RNA Shield). The guidelines below _do not address_ how to process these types of samples. Refer to the manufacturer's protocols for how to process samples for your RNA isolation procedure and tissue preservation method(s). ### Liquid Nitrogen (LN2) with Mortar & Pestle #### Pros - Best method for complete tissue pulverization. - Best method for sample preservation (sample never thaws). - Can be easily adapted for most tissue amounts. #### Cons - Very labor intensive. - Very time consuming. - Requires LN2 which is not readily available in our lab. ##### Example Procedure ::: {.callout-note} Prior to beginning, you'll need to get LN2 from UW Chemistry Stores in Bagley Hall. ::: 1. Pour liquid nitrogen into a small(ish) polystyrene container (or, an ice bucket works). 2. Use 50mL conical to transfer LN2 to a clean mortar (this tube can just be stored in the LN2 for the duration of this entire process). This pre-chills the mortar and is important! 3. While the mortar is pre-chilling, put bottom portion of pestle into LN2 in the polystyrene container to pre-chill. Not too deep, though, or else the pestle handle will get too cold to hold onto. 4. While the mortar and pestle are pre-chilling, place rounded end of steel spatula into LN2 in the polystyrene container to pre-chill. 5. Transfer tissue to pre-chilled mortar. 6. Quickly pulverize tissue using pre-chilled pestle. 7. Transfer powdered tissue, using pre-chilled spatula, to storage tube (preferably pre-chilled in LN2) and/or subset to pre-chilled, zeroed-out tube on scale for weighing. 8. Proceed to next sample with different clean mortar/pestle/spatula. 9. After all mortar/pestles/spatulas have been used, wash with soap and water (use a sponge to wash). 10. Submerge all utensils in a 10% bleach solution for at least 10mins. 11. Rinse all utensils thoroughly with DI water. Dry them thoroughly with paper towels before proceeding. Residual water will be annoying to deal with and could inhibit dowstream processes. ::: {.callout-tip} For more thorough (and faster) drying, use a squirt bottle filled with methanol to rinse all utensils. Do this over a _LABELLED_ plastic "shoebox" container to collect the rinse. Set utensils aside on paper towels for a few minutes. **IMPORTANT**: This should ONLY be done in the fume hood with proper PPE (e.g. gloves). Be sure to double check that no ignition sources are present! Methanol is extremely flammable. Allow collected methanol rinse to evaporate in fume hood. ::: ### Disposable Plastic Mortar & Pestle #### Pros - Supplies are always on hand. - Good for small tissue amounts. #### Cons - Somewhat labor intensive. - Somewhat time consuming. - Tissue might thaw during process. - Sometimes requires dry ice. - Only good for small tissue amounts. ##### Example Procedure ::: {.callout-note} If processing more than 2-3 samples, get dry ice from Biochemistry Stores (Health Sciences, J-wing basement) to prevent samples from thawing while waiting to be homogenized. ::: 1. Fill disposable mortar tubes with homogenization solution (e.g. Trizol, Tri-reagent, kit homogenization buffer, etc.). ::: {.callout-important} Do not fill mortar tubes with more than 500uL of solution! Otherwise, displacement from the pestle will cause the tube to overflow. ::: 1. Store frozen samples on ice - preferably dry ice if more than 2-3 samples. 2. Using small dissection scissors and/or forceps and/or small steel spatula, chip out a piece of frozen tissue. 3. Weigh tissue with weight boat/paper. 4. Transfer tissue piece to pre-filled mortar tube. 5. Homogenize tissue with disposable pestle. Move the pestle up and down, with twisting motions to homogenize tissue. Do this until no visible chunks of tissue remain. ::: {.callout-tip} If processing many samples, using a [motorized Pestle Pellet](https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/images/150/322/fd097467-314f-4e5f-99f0-a24d956a5bc1/800/fd097467-314f-4e5f-99f0-a24d956a5bc1.jpg) will significantly ease strain on your hand/fingers and make the process faster. Use caution to avoid splashing!! ::: ### Bullet Blender (i.e. bead beater) #### Pros - High throughput (i.e. can process many samples simultaneously) - Least lab intensive of all methods. - Can handle almost all tissues sizes - very small to relatively large. #### Cons - Tissue will thaw during process. - Requires dry ice. - Very loud. ##### Example Procedure Review the [Bullet Blender protocol in the Roberts Lab Handbook](https://robertslab.github.io/resources/protocols/protocol-bullet_blender/) before starting. ::: {.callout-note} The Bullet Blender will require dry ice to keep it cool during your run(s). Get dry ice from Biochemistry Stores (Health Sciences, J-wing basement). ::: 1. Pre-fill tubes with desired beads and homogenization buffer. ::: {.callout-tip} Prepare tubes a day or two ahead of time to save you time on the actual day you perform the extractions. ::: 2. Store frozen samples on ice - preferably dry ice if more than 2-3 samples. 2. Using small dissection scissors and/or forceps and/or small steel spatula, chip out a piece of frozen tissue. 3. Weigh tissue with weight boat/paper. 4. Transfer tissue piece to pre-filled tube. 5. Pre-chill Bullet Blender. 6. Run Bullet Blender with desired time/intensity.