Can I use OCT embedded fresh frozen tissue with the Flex/Fixed RNA Profiling assay?

Question: Can I use OCT embedded fresh frozen tissue with the Flex/Fixed RNA Profiling assay?

 

Answer: Based on limited testing, we have successfully generated Single Cell Gene Expression Flex (i.e., Fixed RNA Profiling) libraries from OCT embedded fresh frozen tissue using either a modified version of the Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling Demonstrated Protocol (detailed below) or the Chromium Nuclei Isolation Kit.  

 

The modified version of the Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling Demonstrated Protocol outlined in this article is the preferred sample preparation method for OCT embedded fresh frozen tissue for use in the Flex/Fixed RNA Profiling assay.  If the Chromium Nuclei Isolation Kit is used, significantly more sections/curls will be required due to variability in nuclei yield.  Data from limited testing from both sample preparation methods from the same OCT blocks are shared in this article.

 

Modification of the Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling Demonstrated Protocol for OCT embedded fresh frozen tissue 

Chromium Next GEM RNA Profiling Sample Fixation Kit, 16 rxns (PN-1000414) will be needed for tissue fixation.  To dissociate OCT embedded fresh frozen tissue for Flex, please follow the modified version of the Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling Demonstrated Protocol outlined below for sample fixation.  2 x 25 µm OCT curls  were used for each sample tested in this modified protocol. There is no additional guidance for this method outside of this article.

 

Fix Tissue:

1. 2 x 25 µm OCT curls were obtained from histology core. The OCT sections were trimmed to minimize excess OCT carryover.  The curls were placed in 1.5 mL tubes. 
2. Fix the curls in 1.5 mL tubes for 16-24 h at 4°C using 1 ml Fixation Buffer reagent. Upon adding the Fixation Buffer, some of the curls might become gelatin-like and stick to the side of the 1.5 mL tubes. (Do not attempt to detach the curls from the tubes. Due to the sticky nature of the curls, they may stick to pipette tips resulting in sample loss.)
3. Centrifuge at 850 rcf for 5 min at room temperature.
4. Remove the supernatant without disturbing the tissue pellet, leaving roughly 100 µl of supernatant to not disrupt the OCT pellets. 
5. Add 1 mL of chilled PBS. The curls came off the side of the tubes but did not break apart and remained intact. The curls were collected by centrifuging at 850 rcf for 5 min at room temperature.
6. Remove the supernatant without disturbing the tissue pellet, leaving roughly 100 µl of supernatant to not disrupt the OCT pellets. 
7. Repeat the wash step one more time for a total of two washes.
8. Add 1 ml of chilled Tissue Resuspension Buffer or Quenching Buffer.  The curls were incubated on ice for 10 min.
9. Centrifuge at 850 rcf for 5 min at room temperature.

 

Dissociation of fixed OCT curls using Octo Dissociator:

1. Remove Quenching without breaking the curls.
2. Add 1 ml of Dissociation Solution. Please see  Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling Demonstrated Protocol for additional details.  Transfer to Miltenyi C tubes. 
3. Rinse the 1.5 mL tubes with an additional 1 ml of Dissociation Solution.  Add to respective Miltenyi C tubes.
4. Place the C tube in the Octo Dissociator, apply Heating Units, and run the following program: 

5. Centrifuge C tubes at 300 rcf for 5 min at room temperature to collect dissociated cells at the bottom of the tube. Resuspend the pellet in the supernatant.
6. If tissue chucks/flecks  are still present in the suspension, continue to pipette mix until they are broken apart prior to filtering the suspension.  The tissue chucks/flecks may clog the filter.
7. Pass the suspension through a Pre-Separation Filter (30 µm) placed on a 15-ml tube at room temperature.
8. Rinse the 30 µm with 2 mL of PBS to recover the cells from the filter.  Collect the filtrate in the same tube as the previous step.
9. Centrifuge the cell suspension at 850 rcf at room temperature for 5 min.
10.  Resuspend the pellet in 1 mL chilled Tissue Resuspension Buffer or Quenching Buffer, pipette mix 5x, and maintain on ice. 
11.  Determine cell concentration of the fixed sample using an Automated Cell Counter. For accurate cell counting, it is strongly recommended that the cell suspension be stained with a fluorescent nucleic acid dye such as Ethidium Homodimer-1 or PI Staining Solution and counted using an automated fluorescent cell counter.
12. Proceed immediately to appropriate Chromium Fixed RNA Profiling protocols – Probe Hybridization step 1.1d.

Manual Dissociation (This has not been tested in-house for this sample type, but guidance is provided below to enable customer success):

1. Remove Quenching without breaking the curls.
2. Add 1 ml of Dissociation Solution.  Please see  Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling Demonstrated Protocol for additional details. 
3. Incubate for 20 min at 37°C shaking the tube intermittently. Using a P1000 pipette tip or a silanized glass pipette, triturate the tissue pieces 15-20X (until solution begins to turn cloudy) to obtain a single cell suspension.
4. Pass the suspension through a Pre-Separation Filter (30 µm) placed on a 15-ml tube at room temperature.
5. Rinse the 30 µm filter with 2 mL of PBS to recover the cells from the filter.  Collect the filtrate in the same tube as the previous step.
6. Centrifuge the cell suspension at 850 rcf at room temperature for 5 min.
7.  Resuspend the pellet in 1 mL chilled Tissue Resuspension Buffer or Quenching Buffer, pipette mix 5x, and maintain on ice. 
8.  Determine cell concentration of the fixed sample using an Automated Cell Counter. For accurate cell counting, it is strongly recommended that the cell suspension be stained with a fluorescent nucleic acid dye such as Ethidium Homodimer-1 or PI Staining Solution and counted using an automated fluorescent cell counter.
9. Proceed immediately to appropriate Chromium Fixed RNA Profiling protocols – Probe Hybridization step 1.1d.

 

Please note that the tissue density in combination with tissue cross section area (tissue volume) will impact final cell yields.  If lower than expected cell yields are obtained, this sample preparation protocol may need to be repeated to increase yield.

 

Sample Storage Recommendations:

While this has not been tested in-house, it may be possible to store fixed and dissociated cells resuspended in Quenching Buffer following the recommendations in the  Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling Demonstrated Protocol.  No additional guidance for this modified protocol is available outside of this article.

 

Table 1: Cell Yields from OCT embedded Fresh Frozen Tissue Sections from Human Tissue using modified Tissue Fixation and Dissociation DP

Table 2: Summary of Gene Expression Flex data generated from samples isolated from OCT embedded Fresh Frozen Human Tissue Sections using modified Tissue Fixation and Dissociation DP*

 

*10k cells were loaded per barcode (~40k cells loaded/well), with a targeted recovery of ~6k  per barcode.  The liver sample was sub pooled across BC002/BC003.  

 

Figure 1: Barcode Rank Plots and tSNE plots for samples isolated from OCT embedded Fresh Frozen Tissue Sections from Human Tissue using modified Tissue Fixation and Dissociation DP

 

Together, these data illustrate that it is possible to generate Single Cell Gene Expression Flex data from OCT embedded Fresh Frozen tissue.

 

Please note: This modified protocol has only been tested in a limited capacity for OCT embedded fresh frozen tissue.  A pilot experiment is recommended for your specific tissue type of interest prior to committing to a larger study.  We have not tested OCT embedded fixed frozen tissue with or without sucrose cryoprotection.  If using OCT embedded fixed frozen tissue, we recommend proceeding directly to tissue dissociation. Please note that this guidance has not been tested on fixed frozen tissue and a pilot experiment is recommended.

 

Nuclei Isolation from OCT embedded fresh frozen tissue using Chromium Nuclei Isolation Kit

The modified Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling Demonstrated Protocol outlined above is the preferred sample preparation method for this tissue type for use with Flex, due to the significant number of tissue sections needed and the variability in yield using the  Chromium Nuclei Isolation Kit. If the Chromium Nuclei Isolation Kit (CNIK) is used, significantly more sections/curls will be required. Summarized below are data generated during limited testing in-house from three Human OCT embedded Fresh Frozen Tissues using  the Chromium Nuclei Isolation Kit.

 

Table 3: Nuclei Yields from OCT embedded Fresh Frozen Tissue Sections from Human Tissue using CNIK

 

Table 4: Summary of Gene Expression Flex data generated from nuclei isolated from OCT embedded Fresh Frozen Tissue Sections from Human Tissue using CNIK**

 

**10k nuclei were loaded for the kidney and liver samples, with a targeted recovery of ~6k nuclei for the kidney and liver samples; ~500 nuclei were loaded for the brain sample. Due to the low nuclei yield for the brain sample, all of the post-hyb sample was pooled with the kidney and liver samples.

 

Figure 2: Barcode Rank Plots and tSNE plots for nuclei isolated from OCT embedded Fresh Frozen Tissue Sections from Human Tissue using CNIK.

 

**10k nuclei were loaded for the kidney and liver samples, with a targeted recovery of ~6k nuclei for the kidney and liver samples; ~500 nuclei were loaded for the brain sample. Due to the low nuclei yield for the brain sample, all of the post-hyb sample was pooled with the kidney and liver samples.

 

Best Practices when working with OCT embedded fresh frozen tissues:

• Please review Visium Spatial Protocols – Tissue Preparation Guide for additional guidance for  proper tissue handling and preparation techniques to preserve the morphological quality of the tissue sections and the integrity of mRNA transcripts.
• Measuring the RIN score of the tissue block is recommended when the block quality is unknown or compromised.  It is recommended to assess RNA quality of the tissue block at this stage by calculating RNA Integrity Number (RIN) of freshly collected tissue sections. RIN should be ≥ 7. Various factors could lead to low RIN scores, such as specific tissue types, diseased or necrotic tissues, sample preparation and handling.
• To maintain RNA integrity, minimize freeze-thaw and sectioning cycles before isolating cells from OCT curls.

 

References 

1. Chromium Fixed RNA Profiling Reagent Kit for Singleplexed Samples with Feature Barcode technology for Cell Surface Protein User Guide (CG000477) 

2. Chromium Fixed RNA Profiling Reagent Kit for Multiplexed Samples User Guide (CG000527)

 

Products: Fixed RNA Profiling, Single Cell Gene Expression Flex