Objective: Take the tissue samples I collected from the sea pens earlier for sequencing (using as a check to compare to other samples to see if the sea pens may have given different data earlier since the longer they were in captivity the more stress they were under) and the daytime and nighttime tissue samples and extract RNA from them Using Roberts Lab RNA isolation protocol, then create CDNA using the Roberts lab Reverse Transcription protocol .(https://github.com/sr320/LabDocs/wiki/Common-Lab-Protocols) 

RNA extraction

1. Read the SOPs for each of the reagents used in this protocol.
2. Read the manufacturer's RNAzol RT protocol for Total RNA Isolation.
3. Read this protocol.
4. Verify sufficient quantities/availability of reagents/equipment.
5. Wear clean gloves and change gloves frequently.
6. Aliquot 500uL of RNAzol RT to pestle tubes and store on ice.
7. Transfer tissue to pestle tubes containing RNAzol RT.
8. Homogenize immediately with disposable pestle.
9. Immediately add additional 500uL of RNAzol RT to pestle tube.
10. Vortex 15s.
11. Add 400uL of 0.1% DEPC-treated H2O.
12. Vortex 15s.
13. Incubate at room temperature (RT) for 15mins.
14. Centrifuge 12,000g for 15mins @ RT.
15. Transfer 750uL of supernatant (do not disturb pellet) to sterile 1.7mL snap-cap tube. (Discard remaining liquid in RNAzol RT Hazardous Waste container in fume hood. Leave old tube open in fume hood over night and then discard in regular trash.)
16. Add 1 volume of isopropanol.
17. Vortex 5s.
18. Incubate @ RT for 15mins.
19. Centrifuge 12,000g for 10mins @ RT.
20. Discard supernatant; do not disturb pellet.
21. Add 400uL of 75% ethanol, centrifuge 4,000g for 3mins @ RT.
22. Repeat steps 20 and 21 one time.
23. Repeat step 20.
24. Centrifuge 4,000g for 1min @ RT.
25. Removal residual ethanol.
26. Immediately resuspend pellet in appropriate volume of 0.1% DEPC-H2O (volume is dependent upon pellet size, but 50uL is usually sufficient).
27. Keep sample on ice for short-term storage (i.e. no more than 2hrs) or store @ -80C.

Reverse Transcription

Standard Operating Protocol (SOP)

Written 20150702 by Sam White.

Reagents:

• M-MLV Reverse Transcriptase (Promega: M1701)
• Primers (oligo dT: Promega: C1101 OR random: Promega: C1181)
• 10mM dNTPs (Promega: U1511)

Personal Protective Equipment (PPE):

• Gloves

Equipment:

• Pipettes (10 - 1000uL)
• Filtered pipette tips
• 0.5mL snap-cap microfuge tubes (Genesee: 22-178A)
• Sterile 1.7mL snap-cap microfuge tubes (Genesee: 22-281S)
• Thermal cycler, water bath, or heating block capable of 37C OR 42C.
• vortexer
• ice

Procedure

Total Time: ~ 1.5 - 2.0hrs

Cost/sample: ~ $1.50

IMPORTANT: A single reaction volume = 25uL. The volume of RNA, primer(s) and M-MLV RT used in this protocol are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA. You may need to make changes to accommodate your own conditions.

1. Read the manufacturer's protocol.
2. Read this protocol.
3. Verify sufficient quantities of reagents and samples before beginning.
4. Wear clean gloves.
5. Thaw all RNA and reagents on ice. Prepare all reactions on ice.
6. Transfer 1ug of RNA to 0.5mL snap cap tubes or PCR plate. Adjust volumes of individual samples to 17.75uL with H2O.
7. Add 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT Cat#C1101 in this example). Total volume (RNA + primers) should equal 18.25uL.
8. Heat samples at 70C for 5 min in thermal cycler, heating block, or water bath.
9. Place samples on ice IMMEDIATELY.
10. Make Master Mix:
    Per Reaction
    • 5 uL 5x Buffer (M-MLV RT Buffer)
    • 1.25 uL 10mM dNTPs
    • 0.5 uL M-MLV RT per ug of RNA
11. Mix well by flicking; do not vortex.
12. Add 6.75uL of master mix to each reaction.
13. Mix by pipetting; do not vortex.
14. Incubate @ 42C for 1hr for oligo dT primers OR @ 37C for random primers.
15. Heat inactivate @ 95C for 3 min.
16. Spot spin and store @-20C.

CDNA Calculations

Results
Sucessfully extracted RNA from the tissue samples and checked the RNA concentrations of all samples using the NANO drop (ND-1000) shown below, all RNA extractions were successful.
(note: I thought it was interesting that all of the day time samples had a higher concentration than the night time samples though I can not draw any conclusions from it an interesting find)
CDNA was sucessfull the next step will be to run a QPCR to check expression levels. 


RNA Concentrations ng/ul
1-1=916.4
1-2=423.0
2-1=934.9
2-2=737.0
Day-1=322.9
Day-2=412.4
Day-3=403.1
Day-4=729.1
Night-1=152.2
Night-2=314.2
Night-3=276.7
Night-4=202.4