RNA QC Saga

Author

Norah Jones

Published

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1 NEB Total vs Qiagen, 2022

Note

Note that the experiments in this section all need to be redone.

1.1 Comparison by Kits

Figure 1

1.2 Comparison by PMD

Figure 2

2 Light-Seq preparation of Gerbil Sections

The first run of LS that we have attempted was produced using OCT section of gerbil tissue. These were freshly prepared by following the Light-Seq protocol and making slight changes to the the fixation protocol as well as the percentage of sucrose incorporated into the OCT embedding medium. These from which we sampled the RNA quality were processed through the entirety of the Light-Seq protocol. The readouts that we were looking for were, RNA quality prior to reverse transcription, quality of cDNA after reverse transcription and quality of cDNA library assayed by qPCR.

2.1 RNA Quality

2.1.1 TapeStation Results

Samples were sent to ULCG for a TapeStation assay. This assays provided the quality metrics for RNA extracted from each section of the conditions assayed Figure 3.

Conditions
Condition 1 2 3 4 Cryoprotectant solution
OCT 4% PFA, 0.25% Triton-X 100, 25 mins PBS Wash x3 30% Sucrose o/n 1:1 OCT:30, 3 hours Pure OCT
OCT-15.1 4% PFA, 0.25% Triton-X 100, 25 mins PBS Wash x3 30% Sucrose o/n 1:1 OCT:30, 3 hours 1:1 OCT:30 [15% sucrose]
OCT-15.2 4% PFA, 0.25% Triton-X 100, 25 mins PBS Wash x3 7% sucrose, 10 mins 1:1 OCT:30 [15% sucrose]
OCT20 4% PFA, 0.25% Triton-X 100, 25 mins PBS Wash x3 7% sucrose, 10 mins 1:1 OCT:30 [20% sucrose]

Concentration.

Gerbil eyes embedded in 100% OCT provided a section that was demonstrably easier to extract a larger volume of RNA, the highest in this experiment, 2.6 ng/µL.

DV200

DV200% was generally quite poor, the highest 2 were obtained from 15% sucrose:OCT sections, indicating that possibly sucrose helps to improve the quality of RNA int he sections but not necessarily in the overall amount of RNA.

RIN

RNA was very fragmented in all sections, no sectioned obtained a score of 4 or higher indicating that the sections have very poor storage of RNA and the same RNA is very fragmented, largely under 200 bps.

Figure 3: RNA Quality of Gerbil OCT sections

2.1.2 Gel Electrophoresis

After in situ Reverse Transcription (in situ RT) the sectioned were barcoded as per the LS protocol. Afterwards cDNA was released from the LS sections and subsequently HPRT1 and Rhodopsin were purified using MegaMix Blue mastermix. The primers for the two targets of interest were engineered to span an exon-exon boundary so that amplification would indicate the success of cDNA synthesis Table 1. Gel run was 1% Agarose stained with SYYBR Gold nucleic gel stain.

Table 1: Gerbil Housekeeping primers
Target Forward Reverse
Rhodopsin (Gerbil) 5’CTTCGGAGAGAACCATGCCA 5’AGTCGATCCCACATGAGCAC
HPRT1 (Gerbil) 5’TTACGGCTTTCCTGGAGGTG3 5’CATCGCCAATCACGATGCTG

Despite the stain not demonstrating clear amplification of our targets there is a visible smear, approximately at the size of the Rhodopsin PCR product (150kb) Figure 4. This smear would later be confirmed by qPCR.

Note

this qPCR was run on Rods BioRad Machine so the data isn’t on my computer. Gary has access to the curves. There is a CSV file that I have but needs to be thought off in terms of how to present this.

Figure 4: Amplification of Housekeeping Genes from LS derived cDNA

3 Comparing FFPE vs OCT RNA isolation

With intent to characterise the RNA quality that is possible to isolate from fixed human tissue, we began by freshly dissecting and embedding human tissue in OCT and FFPE (paraffin). Sections of appropriate size, typical to the kind of experiments that are usually done with these sections were collected and RNA was isolated using the Qiagen RNeasy FFPE kit. Importantly we were interested in how FFPE compares with OCT when it comes to RNA quality as well as how does the number of sections impact the quality and concentration of RNA isolated. For this experiments we cut curls (also called rolls) rather than collected the sections onto slides.

For FFPE sections, we took compared across 3 volumes: 2 and 4 x 10µm. For OCT we collected 20, 40 and 60 x 10µm. The quality of RNA isolated is presented in Figure 5.

Note

In both types of embedding a technical repeat was done for FFPE: 4x10µm and for OCT 40x10µm. These are marked with an asterisk.

Figure 5: Quality of RNA, FFPE vs OCT

rRNA ratio

4 FFPE vs OCT RNA isolation - number of sections matched

For this comparison we took the same as as before but importantly we took precaution to section the same amount of tissue for a more direct and comparison.

The way in which we sectioned and used the section is illustrated below, see Figure 6.

Figure 6: Sections matched by number. FFPE vs OCT
Figure 7: FFPE vs OCT number of sections matched

4.1 Reverse Transcription

4.2 Maxima vs HC

Table 2: Main Caption
(a) Light-Seq, Maxima H Minnus
Temp ºC Time
Phase 1 22 30s
Phase 2 8 30s
15 30s
25 1 mins
30 1 mins
37 1 mins
42 2 mins
Phase 3 42 30 mins
4 Hold
(b) High Capacity
Temp ºC Time
Phase 1 25 10 mins
Phase 2 37 120 mins
85 5 mins
Phase 3 4 Hold

4.2.1 Amplification Plot

4.2.2 Melting Curve Plot Analysis

4.3 Troubleshooting the Reverse Transcription programme

insert picture of the modified programme for Maxima, next to the HC programme

4.4 qPCR

insert the tables of the programme

4.5

4.5.1 Amplification plot

4.5.2 No Triton Comparison

FFPE vs OCT

4.5.3 Triton Comparison

FFPE vs OCT

4.5.4 ΔTºC Comparison

FFPE vs OCT

5 Recent FFPE vs OCT isolation