Tissue to C T : Actual qRT-PCR Results from LCM Samples

1. Tissue to CT: Actual qRT-PCR Results from LCM Samples Joel L. “Jock” Moore, Jr. June 24, 2009

2. Tissue Preparation M222 & M224 were mice with normal hearts. Each received intramyocardial injections of hMSCs. • M303 was an infarct model that also received an intramyocardial injection of hMSCs. • Mouse hearts were harvested, embedded in OCT, and snap-frozen in liquid N2. • Hearts were stored > 2 years at -80 °C

3. Slide Preparation • PALM PEN-Membrane slides were sprayed with RNaseZap (Ambion), dipped in DEPC-treated H2O 2X, then allowed to dry. Prior to cutting tissue on the cryostat, all slides were UV-treated for ≥ 30 min. This not only sterilizes the slides but also allows tissue to adhere more easily to the PEN membrane (UV light alters its hydrophobic nature).

4. Slide Preparation • For each animal, multiple 10 μm sections were cut with an RNase-free cryostat blade and placed on the membrane slides. • A few sections were also placed on RNase-free glass slides to serve as pre-staining RNA quality control samples. • Cost saving • More difficult • Slides were placed back-to-back in a Falcon 50ml conical tube, then stored at -80 °C for a few days.

5. Staining Protocol: General Tips • Overall, the dryer and colder your tissue sections are, the less likely ribonuclease activity will degrade your RNA • OCT must be removed before microdissection • Keep all reagents ICE-cold • Limit aqueous phase staining steps • End your staining protocol with a dehydration step e.g., 100% EtOH (NOT xylenes). This will allow you to work longer on the LCM system with minimal RNA degradation

6. Example H&E Staining Protocol • Retrieved Falcon tube from freezer and let thaw (~ 5 min). • “Fixed” membrane slide in 70% EtOH, 2 ½ min. In the meantime, dipped the glass slide in DEPC-treated H2O then scraped tissue directly into a microfuge tube containing lysis buffer. • Hematoxylin QS (Vector Labs), 30 sec. • Dip in DEPC-treated H2O • Eosin-Y, few seconds. • Quick increasing EtOH series (70%, 96%, 100%). • Removed excess and let dry on Robomover stage. • While drying, used the 5X objective to scan the entire slide.

7. PALMRobo Software Settings • These settings will vary dramatically by objective, embedding material, tissue type, even staining protocol. • My settings using a 10X objective • RoboLPC joint = 16 μm • Cut speed = 50 μm/sec • Cut/LPC Focus Δ = - 3 (Cut: 87, LPC: 84) • Cut/LPC Energy Δ = +40 (Cut: 52, LPC: 92) • Fine-tune & save your settings before performing experiments on precious tissue samples.

8. M224 Injection Site

9. M224: Injection Site, Adhesive Cap

10. M303 Infarct Site

11. M303: Infarct Site, Adhesive Cap

12. M303: Normal Myocardium

13. M303: Normal Myocardium, Adhesive Cap

14. Examples of LPC Elements • M303 • Infarct site: 20 Circular elements @ ~ 38,000 μm2each (~ 760,000 μm2 total) • Normal myocardium: 9 circular elements @ ~ 67,000 μm2 each (~ 600,000 μm2 total) • M224 • Injection site: 4 elements of varying size (~ 128,000 μm2 total) • Normal myocardium: 6 circular elements @ ~ 37,000 μm2 each (~ 225,000 μm2 total)

15. 5X Scan, M303 Post-LMPC * Unsuccessful LPC-elements

16. RNA Preservation & Extraction • I used QIAGEN’s RNeasy Plus Micro Kit (Cat # 74034) • Small elution volume of 12 μl • Ambion’s RNAqueous-Micro Kit (Cat # AM1931) is another popular choice. • Because ß-mercaptoethanol permanently denatures RNases and lysis buffers are designed to stabilize RNA, you can relax once your sample is submerged in this solution!

17. RNA Characterization • Agilent 2100 Bioanalyzer (RNA Pico 6000 Lab-on-a-Chip Kit) • This system uses microfluidics technology to perform an electrophoretic analysis of RNA using only 1 µL of an RNA sample. • Its software generates an electropherogram, a virtual gel-image, and a quantitation estimate. • It is qualitatively accurate at [RNA] between 50 – 5000 pg/μl, but its quantitation accuracy is reported as 30% CV (Coefficient of Variance = SD/mean).

18. Agilent 2100 Bioanalyzer • The RNA integrity number (RIN) is a value automatically assigned to total RNA samples by the Bioanalyzer’s software. Rather than using a ratio of ribosomal bands, the software uses the entire electrophoretic trace to identify the 28S &18S ribosomal peaks while also taking into account the presence or absence of degradation products. Agilent touts the system as the de facto standard for RNA integrity since the assigned RIN is independent of sample concentration, instrument and analyst. The RIN Continuum Maximum degradation Lowest RNA quality Minimum degradation Highest RNA quality 1 10

19. Agilent 2100 Bioanalyzer • What the RIN cando: • Obtain a numerical assessment of the integrity of RNA. • Directly compare RNA samples, e.g. before and after archiving, or compare integrity of the same tissue across different labs. • Ensure repeatability of experiments, e.g. if RIN shows a given value and is suitable for microarray experiments, then the RIN of the same value can always be used for similar experiments given that the same organism/tissue/extraction method is used. • What the RIN cannot do: • Tell a scientist ahead of time whether an experiment will work or not if no prior validation was done (e.g. RIN of 5 might not work for microarray experiments, but might work well for an appropriate RT-PCR experiment. Also, a RIN that might be good for a 3' amplification might not work for a 5' amplification) *Directly from Agilent’s website

20. M222 RNA Degradation: ~40 min post thaw RIN: 8.4 RIN: 7.6

21. RIN: 8.3 We could not locate M222’s injection site on this particular membrane slide. The normal myocardium LCM sample, however, looks to be of sufficient RNA quality.

22. M224 RNA Degradation: ~40 min post thaw RIN: 8.8 RIN: 6.9

23. RIN: 9.5 M224’s Possible Injection Site LCM sample yielded high quality RNA

24. RIN: 7.9 M224’s Normal Myocardium LCM sample also showed promise.

25. M303 RNA Degradation: ~40 min post thaw RIN: 8.9 RIN: 6.8

26. M303 Normal Myocardium LCM sample should produce qRT-PCR results, but… RIN: 8.6

27. The M303 Infarct LCM sample is not looking good. RIN: 1.0

28. RNA Quantitation • Agilent • Nanodrop

29. Nanodrop Spectra

30. qRT-PCR • cDNA was synthesized using Invitrogen’s SuperScript First Strand Synthesis system • Made 10 ng cDNA based on Nanodrop figures • Used Taqman Probes to search for Human GAPDH and Mouse GAPDH. This will answer the question, “Are these human or mouse cells?”

31. qRT-PCR Results *CT cut-off set at 40 cycles

32. Changes for future experiments Quantitation of RNA? Amplification of RNA? Pre-amplification of cDNA? Quantitation of cDNA? (Standard curves, Picogreen fluorometric assay, etc.)

33. http://www.ecu.edu/cs-dhs/internalmed/ec/lasercapture.cfm

34. Thanks! Email me at moorejo@ecu.edu Or call 4-5024 with any questions