Genomics/Microarray Facility

Helpful Information

Explanation of Quality Measures
Microarray Facility Protocols (Denaturation of Filter Arrays)
RNA Extraction
DNAse Treatment of RNA
Labeling RNA Targets
A: Oligo dT labeling
B: Pre-Hybridization
C: Washing Filters
D: Stripping Filters for Reuse
Hybridization Schematic
Batch hybridization 2 filter
mRNA Amplification
Real Time PCR
Imagene Instructions

RNA Extraction
Reprinted from Ambion's TechNotes Newsletter 3:3, © 1998
One of the most problematic steps in RNA isolation is the first step - thorough lysis of the tissue or cell sample in a denaturant solution that inhibits RNA degradation by RNase. While it is possible to process fresh tissue directly, it is extremely important that all cells are disrupted immediately upon contact with the denaturant. This usually requires use of a polytron and even then some "difficult to process tissues" (e.g. hard tumors, bacterial cells, plant roots, etc.) are not effectively disrupted (see the article, "Isolating Intact RNA"). Therefore, if you are having a problem with yield or degradation during RNA isolation, we usually recommend freezing the tissue sample before processing. Here we compare three methods for processing frozen tissues in a side-by-side test for the quantity of mRNA recovered.

Freezing the Tissue 
Samples should be frozen quickly so that the whole tissue sample freezes at once throughout. This may mean mincing the tissue into smaller fragments before freezing. Submerging the samples in liquid nitrogen will freeze the tissue pieces most quickly. Alternatively, a metal plate placed on dry ice can serve as a freezing surface.

Each of the methods below describes a distinct way of generating a tissue/cell lysate from which to purify RNA and is assessed for yield of poly (A+) RNA, when used to process 0.1 g of frozen mouse liver tissue. While the three methods each use a guanidine buffer to ultimately lyse the cells, they differ in how the tissue is processed prior or during that lysis step.

Method 1: Processing Frozen Tissue Fragments in a Dounce 
yield: 4.1 µg poly (A+) RNA 
Frozen tissue is cut into small pieces (approx. 0.5 cm2) on dry ice, placed in a dounce, and processed as lysis buffer is added. Both pestle A and pestle B are used for ten strokes each.

Method 2: Processing Frozen Tissue Fragments through a Syringe 
yield: 3.2 µg poly (A+) RNA 
Frozen tissue is cut into small pieces (approx. 0.5 cm2) on dry ice, added to lysis buffer and passed back and forth ten times through an 18 gauge syringe needle.

Method 3: Grinding the Tissue to a Powder in Liquid N2
yield: 7.1 µg poly (A+) RNA
The frozen sample is powdered by, grinding the frozen tissue fragments in a pre-chilled mortar, and occasionally adding liquid N2 into the mortar to prevent thawing. Once the tissue is ground to a fine powder, the denaturing solution is added to the mortar, and the semi-frozen mixture is stirred. This mixture can then be thawed and transferred to an appropriate vessel for further processing.

Note that by grinding the tissue to a powder in liquid N2 (Method 3), cellular disruption is much more complete resulting almost twice the yield of the other two methods.

Several different procedures for RNA extraction including, TriReagent, (Molecular Research Center) RNAqueous (Ambion) and RNeazy (Qiagen). If you are presently using a procedure that works for you continue to use it. We routinely use Tri-reagent (MRC cat#: TR-118 ) or it can be ordered from the Cell Center). Also order BCP* instead of Chloroform: (MRC cat: BP151).*BCP is less toxic than chloroform and reduces DNA contamination. Tri-reagent gives consistently good yields with little DNA contamination. Follow the manufacturer directions as to the relative amounts of cells and reagent to use taking into account that very small cells (e.g. T and B cells) require less reagent per 106 cells. If the expected amounts of RNA are low we add polyacryl carrier (MRC cat # PC152) to the Tri-reagent at the level of 10ug carrier/ ml of Tri-reagent (see manufacturers directions). For extraction of RNA from cells, the syringe procedure for dispersing the cells and breaking up the DNA works well. For tissue fragments, we always quick freeze first, add Tri-reagent and then use a polytron to break up the tissue in the Tri-Reagent. Be careful not to freeze the polytron. RNAqueos also gives good quality RNA but yields are affected if the DNA is not sufficiently sheared and we find that DNA contamination is always high with this procedure. In addition, the columns frequently clog during the purification due to the presence of DNA/debris clots. No matter how clean the RNA looks on a gel, DNA must be removed by DNAse treatment before the total RNA is labeled and used to probe the arrays.

To reduce the amount of Reagent necessary to extract RNA from attached tissue culture cells, they can be removed from the plate (using Versene or trypsin) washed once with medium +FBS and once with PBS. Resuspend pellet slightly by tapping with finger. Add Tri-reagent to pellet at amounts indicated for the number of pelleted cells.

*Remember to reduce amount of TriReagent for smaller cell pellets.
**When processing large samples (tissues, organs) do not try to reduce the amount of Tri-reagent without considering the following: the Guanidinium concentration must be kept at 6M to inactivate RNAse, to maintain this concentration the sample volume must be less than 10% of the volume of TriReagent added. (back to top)

DNAse Treatment of RNA

All reagents are made in DEPC water and autoclaved or purchased as RNAse  free
*This reaction can be scaled down for smaller quantities of RNA

1. 100 uL RNA (1mg/ml)
   20 uL 10x DNase I buffer
   10 uL DNase (1unit/uL)
   1 uL RNasin
   + 69 uL DEPC water 
   200 uL (100ug RNA per 200uL)
2. Incubate at 37 C for 30 min
3. Add 20uL 0.1M EDTA pH 8.0 and 1uL polyacryl carrier (per how many uL reaction and how much RNA- 100ug does not need carrier).
4. Add equal volume phenol/chloroform/isoamyl alcohol. Vortex, microfuge 5min, remove aqueous phase to a clean eppendorf tube.
5. Add equal volume chloroform to the aqueous phase. Vortex, spin 5' in microfuge, remove aqueous phase to fresh tube.
6. Add 1/10 volume 2M NaOAc and 2.5 vol. 95% EtOH.
7. Vortex, put into dry ice+ ETOH bath for at least 10' (until frozen), microfuge 15'. Remove EtOH.
8. Add 500uL 75% cold EtOH.
9. Microfuge 10'. Remove supernatant.
10. Air dry- do not speedvac. Resuspend in DEPC H2O to bring to ~1mg/mL. (Assuming at least 20%-50% loss of starting RNA). Heat at 50C for 30 min to facilitate resuspension of RNA. If pellet is too dry it is difficult to resuspend.

*For RNA samples with a lot of DNA contamination you may increase DNAse concentration 1.5 -2.0 fold keeping everything else constant.
*Very small amounts of RNA that need to be amplified (see RNA amplification section) are frequently not DNAse treated before amplification.
(back to top)

Denaturation of Filter Arrays

Facility produced arrays are not denatured prior to sale unless specifically ordered as such. The denaturation steps are simple and there is no reason to pay us to do it.

Denaturing Solution
0.5M NaOH, 1.5M NaCl To make 500mLs:
10N NaOH 25mL
5M NaCl 150mL
dH20 325mL

Neutralizing Solution
0.5M Tris-HCl pH 7.8, 1.5M NaCl To make 500mLs:
1M Tris-Cl 250mL
5M NaCl 150mL
dH20 100mL

• Set up two plastic trays with filter paper to size. Soak filter paper in tray 1 with Denaturing solution and dump off excess solution. Soak filter paper in tray 2 with Neutralizing solution and dump off excess solution. Make up a final solution of 0.25x Neutralizing solution in a plastic/metal tub.
• Place filters, DNA side up, onto the denaturing solution/tray 1 for 10 minutes only! Be consistent in the way denaturation is done each time.
• Transfer filters, DNA side up, to the neutralizing solution/tray 2 for 10 minutes (it is okay if the filters remain on neutralizing solution/tray 2 longer).
• Transfer filters to a 10mM Tris bath, pH 7.8 (or 0.25x neutralizing solution bath). The filters can sit in the bath for however long it takes to process all the filters.
• Blot filters from bath onto dry filter paper to remove excess liquid (can let sit a minute to make sure all excess liquid has soaked into dry filter paper). If filters are either too dry or too wet the UV cross-linking is less efficient, but they must be damp.
• Place filters onto fresh aluminum foil and place into Stratalinker or any commercial UV apparatus (ours is from UVB). The Stratalinker should be set to 1200 Joules. Press start- the UV light will come on and it will count down from 1200 Joules (the UVB takes about 12 seconds).
• Place filters on aluminum foil into 80°C oven for 15 minutes to completely dry before storage.
• Sandwich filters between manufacturers paper or filter paper and place them into a plastic ziploc bag. Label bag with filter information and indicate that they have been denatured and are ready for hybridization. We store our filters at -20C.

Labeling RNA Targets

A: Oligo dT labeling            
Note: The convention is to refer to the labeled cDNA/cRNA as the target and the spotted clones on the array as the probes... (except in England).
Protocol is written for a 15ul reaction but may be doubled in volume if using over 4ug Total RNA. This procedure may be used for Research Genetics and Facility arrays. Clontech arrays require a specific set of primers.

1. 1. Add DNAse treated total RNA (3-4ug), mRNA or aRNA (300-500ng) in a 2uL volume or less. Add 1ug Oligo-dT (2uL at 0.5ug/uL) and 1ul 10x Random Decamers (AMBION #5720G2) to an eppendorf tube.
   Bring volume to 5ul with DEPC treated water.

2. Heat tube at 70C 10 min, chill on ice.
3. Add in order : 
3ul 5x first strand buffer (BRL/ life Tech cat # 18064-014)
0.5ul DTT (0.1M BRL/ life Tech cat # 18064-014)
0.75 uL dNTPs (A, T, G 20mM Pharmacia cat# 27-2035-02)
5 uL 33P dCTP 10Mci/ml- sp. act. 3,000Ci/mmol YOU MUST USE 33P FOR THE ARRAYS PRODUCED BY THIS FACILITY. 32P CAN ONLY BE USED FOR CLONTECH ARRAYS.
0.75 uL reverse transcriptase (200u/ul Superscript II RT BRL)*ADD LAST!!!

4. Incubate at 37C 90 min.

5. After incubation bring labeled target volume to 75ul with 1XTE before loading onto a Micro Bio-Spin Columns- 75ul capacity (Bio-Rad Cat # 732-6002) follow instructions that come with columns. Eluted sample is approximately 75ul. Measure the radioactivity of the entire target by placing the eppendorf tube containing the eluted sample into a scintillation vial and counting in the Tritium channel (Cherenkov counts). Calculate % incorporation against 1uL radioisotope stock brought in 75ul of the same solution and volume as labeled target. Divide the total counts in the target by the total counts in the reaction to get percent incorporation. REMEMBER 33P HAS 1/7 THE ACTIVITY of 32P SO THE COUNTS WILL BE LOW BY COMPARISON.* Incorporation should be at least 30% and preferably 50% of the total counts used for labeling. (If you are labeling aRNA, mRNA, or Clontech probes the level of incorporation may be lower).

6. Denature labeled target for 3 min at 100C then chill on ice until ready to use. Use immediately or within 1-2 days. 
(back to top)

B: Pre-Hybridization
***Start Pre-Hybridization procedure before Oligo-dT labeling if you are pressed for time***
1. Prewash filters in 0.5% SDS that has been heated to 90C. Slip filters into the heated prewash solution and agitate at room temperature for 5 min.
2. Blot prewashed filters on clean filter paper and transfer to hybridization bottles containing prehyb solution 1 and incubate for 1hr at 42C with rotation of 7-10rpms.
3. Remove prehyb solution 1 and replace with prehyb solution 2. You can add the MicroHyb Buffer to the bottle and then add poly dA and Cot-1 into the buffer being sure not to touch the filters. Incubate for at least 2 hrs at 42C. 33P labeled denatured probes may be added directly to prehyb solution 2 and hybridized for 18 hours with rotation.
The hybridization volume used will depend on the size and/or number of arrays and the container size. We use bottles in a hybridization oven but plastic bags can be used as well. Prehyb solution volumes are given for 35mm x 100mm bottles: adjust volumes if using different sized bottles (Use a slightly larger volume of hybridization solution for the Salmon Sperm step and then put the least amount of solution needed to cover and keep filters wet for the poly dA and Cot-11 DNA step so that probe is as concentrated as possible). For very small custom arrays we have used tightly sealed scintillation vials.
Prehyb solution 1
3mLs MicroHyb Buffer
Salmon Sperm (final concentration 1ug/mL)- denatured- boiled 3'.
Prehyb solution 2 
2.5mLs MicroHyb Buffer
Poly dA (final concentration 1ug/mL)
Cot-1 DNA (final concentration 1ug/mL)- denatured- boiled 3'.
(Research Genetics Micro-Hyb solution- HYB250.GF, Mouse Cot-1: BRL- Lifetech cat # 18440-016, Human Cot-1: Cell Center (BRL Lifetech cat # 15279-011) Make sure that Cot-1 DNA and Salmon Sperm have been denatured by boiling for 3min. before adding to prehyb buffer.
You may also use Express-hyb from Clontech if you prefer. The hybridization using Express-hyb is carried out at 65-68C instead of 42C. Read manufacturers instructions if you decide to use this hybridization buffer. Express -hyb is essentially Church buffer that you can prepare yourself if you like. Be consistent with hybridization times we carry out hybridization for 16hr.
Church Buffer
For 1Liter
70g Electrophoretic grade SDS
5g BSA Fraction V 
80ml 0.5M Sodium Phosphate buffer pH 7.2
2ml 0.5M EDTA + DDH2O to 1L

C: Washing Filters
Make sure filters do not dry out- or label will remain on filter permanently. Check the counts before and after each wash step. Want about 1-2 counts/sec, if using an ordinary 32P Geiger counter, (more sensitive counters used for S35 will give higher readings).

1. Remove hybridization fluid and rinse filters with a small amount of 2XSSC/1%SDS and discard.
2. Wash Filters with generous amount of preheated 2XSSC/1%SDS at 50C for 20 minutes at hybridization oven top speed ~17-20rpm - (for 35mm x 100mm bottles use ~40mLs). Don't completely fill bottles. It will prevent good movement of the wash during rotation. Repeat.
3. Wash filters the same amount of 0.5XSSC/1%SDS at 55C for 20 minutes at hybridization oven top speed: (for 35mm x 100mm bottles use ~40mLs). Repeat if counts are still high.
4. An additional high stringency wash step may be necessary if using aRNA or mRNA: 0.1XSSC/0.5%SDS at 55C for 20 minutes.

***Once hybridized, never let filters dry (radioactivity will be permanently fixed to filter)***
Filters are place DNA side up on a moistened filter paper (we use 3xSSC) to prevent drying. Turn filter paper/microarray combination over onto a piece of plastic wrap. Pull on plastic wrap to smooth underneath. Fold two sides of the plastic wrap. Turn back over and with a wet glove smooth out remaining bubbles/wrinkles that are on top of filters. Finish folding and place in PhosphorImager cassette. Remember DNA side is against the screen. For a good hybridization you should see some spots after a few hr exposure. You must scan at 50microns in a Storm PhosphorImager for analysis! If blots look dirty after a few hours exposure, repeat last wash before putting on screen overnight. Typically the arrays are exposed over night and for 4 days.
If using Clontech Express-hyb or Church Buffer:
1. Wash filters hybridized in ExpressHyb or Church buffer at 65-68C for 20 min with continuous agitation in pre-warmed 2xSSC/1%SDS. We do this at top speed in the roller bottles. Check counts. Repeat.
2. Wash at 65-68C for 20 min with continuous agitation in prewarmed 1xSSC, 0.5%SDS.
Check counts. Repeat.
3. Wash at 65-68C for 20 min with continuous agitation in prewarmed 1xSSC, 0.5%SDS.
Check counts. Repeat.

***Be very consistent in the washing protocols for filters that are to be compared. This information and labeling information should be recorded for each filter.***

D: Stripping Filters for Reuse
Filters can be reused 3 to 5X depending how stringently they need to be stripped to remove residual counts. If Using 5X the signals may be diminished..
1. Make ~1-300mL of 0.4M NaOH in a plastic container and heat to 45C in shaking water bath. Once heated, completely submerge filters in the solution and agitate at 45C for 30 minutes.
2. Make ~2-500mL of 0.5% SDS in plastic container and heat to boil in microwave (~3 min). After step 1 is complete, transfer filters to SDS solution, place on rocking platform and agitate at Room Temp for 1hr. Be careful handling the hot SDS.
3. Check with Geiger counter and expose overnight on PhosphorImager screen to make sure it is completely stripped. Stripped filters may be scanned at 200microns because they will not be used for analysis. Sometimes it is necessary to repeat the procedure.
Note: During both washes, the plastic containers should be covered to maintain temperature. Reference: Current Procedures in Molecular Biology Green et al. 2.10.6.
For a more stringent procedure: Strip filters by submerging in 0.1% SDS, 2mM EDTA that has been brought to a boil. Shake at 68C for 30min. 
(back to top)

mRNA Amplification

**PLEASE READ ENTIRE PROTOCOL BEFORE STARTING!!
Frequently it is not possible to isolate the amounts of RNA required for good probe preparation. This will be especially true for arrays using fluorescence that require using mRNA probes. We also find that using message or amplified RNA (aRNA) gives much better signal to noise values even on radioactive arrays. This is especially true for the Research Genetics Filters.
Protocol is to be performed in an RNase/DNase free environment. All reagents used for this procedure are kept separate from general lab solutions and handled only with disposable equipment. Set up a central place in the -20C freezer for all the reagents to be kept together. These reagents are expensive.
1. First strand cDNA synthesis:
In a 0.2ml PCR reaction tube, mix 1ul (1-3ug) total, DNAse treated, RNA with 9ul of a 1.7M trehalose stock solution (saturated, very viscous, warm to 37C to pipette). Add 1ul of the Oligo-dT/T7 primer (stock concentration= 1ug/ul). Heat samples at 65C for 10 minutes then cool/store on ice. 
To each tube add:
4uL 5X First Strand Buffer (comes with Superscript II)
2uL 0.1M DTT (comes with Superscript II)
1uL RNasin
1uL 10mM dNTP mix (Promega Pt#: U1240- A, T, G, and C dNTPs- 100mM)
1uL 0.1ug/uL Linear Acrylamide (Ambion 9520, 5mg/mL)
1uL Superscript II, reverse transcriptase
Using a thermocycler, incubate samples using the following program: 37C for 5minutes > 45C for 5 minutes, then 10 cycles alternating between 60C for 2 minutes > 55C for 2 minutes. When finished, spin briefly and store samples on ice.
2. Second strand synthesis:
To each first strand reaction tube add:
106uL of DEPC water
15uL 10X Second strand buffer
3uL 10mM dNTP mix
1uL E.coli DNA Ligase (10 U/uL) (NEB205L)
4uL E. coli DNA Polymerase (10 U/uL) (NEB 209L or core facility)
1uL RNAse H (2 U/uL)
Incubate for 2 hours at 16C in thermocycler
To stop reaction, add 7.5ul of 1M NaOH/ 2mM EDTA and incubate at 65C for 10 minutes. Transfer sample to nuclease-free 1.5ml microfuge tube.
3. Double-strand cDNA cleanup:
Samples are phenol extracted once using "Phase Lock Gel" tubes from Eppendorf (cat # 0032 005. 055). Read the PhaseLock literature before using.

Pre-spin the Phase Lock tubes for 20 seconds at maximum speed to pellet the gel.

Add 150ul Phenol:Chloroform:Isoamyl alcohol (25:24:1) to cDNA reaction sample from the second stand synthesis above (sec 2). Mix by pipetting 10X then transfer to Phase Lock Gel tubes. Spin for 5 minutes in microfuge at maximum speed, room temperature. You must get 3 phases after centrifugation, if not centrifuge again. Transfer aqueous phase to an RNAse free 1.5ml tube.
To precipitate extracted cDNA, add 70ul 7.5M ammonium acetate (prepared in DEPC water, 0.2um filtered) and 750uL of absolute ethanol (-20C). Vortex, then microfuge for 20 minutes at maximum speed- room temperature. At this point you should see a small salt/polyacryl carrier pellet. Wash pellet with 1mL of 75% ethanol 1 time, spin for 5 minutes. Remove EtOH and allow sample to air dry for a brief time. Resuspend pellet in 40ul DEPC water. You may stop here, store samples at -20C.
Prespin a Chroma Spin DEPCH2O-100 column for each sample at 2400rpm (700 x g), 5 minutes at room temperature using the Sorvall 6000 swinging bucket rotor (or follow manufacturer's instructions). Place spin column into a nuclease-free 1.5ml microfuge tube. Add sample carefully to the center of the column. Centrifuge at 2000 rpm for 5 minutes. Speedvac sample flow-through to 15ul or less.
4.In vitro Transcription:
The Ambion T7 Megascript Kit (#1334) is used for in vitro transcription.
For each sample add:
4uL each 75mM NTP (A, G, C, and UTP)
4uL 10X reaction buffer, thaw and store at room temp. until needed.
4uL T7 enzyme mix
_uL ds cDNA (from step 3)
_uL DEPC water
bring volume up to 40ul using DEPC water.
Incubate for 4 to 6 hours at 37C. Check temperature to be sure it is constant.
Add 1uL DNase I (located in Megascript kit) and incubate for 15 minutes at 37C.
Run 1 uL of sample on RNA quick gel with a size standard.
5. Purification of aRNA from step 4:
Add 40uL Phenol:Chloroform:Isoamyl and mix by pipetting 10X. Phenol extract the samples 1X using the Phase Lock Gel tubes, as instructed in the "cDNA cleanup" section above. The sample is then loaded onto a Clontech Chroma Spin DEPCH2O -30 column. Prespin the column while performing the phenol (phase lock) extraction. Transfer column to a nuclease-free 1.5ml tube and load sample onto the center of the column. Centrifuge (swinging bucket rotor) for 5 minutes at 2400rpm (700 x g). You may wish to run 1 uL out on a 1% agarose gel. *see quick RNA gel protocol Check aRNA concentration by measuring 260/280 OD readings. In most cases this one round of amplification will be sufficient. Approximately 500ng of aRNA is used /hybridization. Yields at this point starting from 3ug total RNA (app. 30ng message) are 5-30ug aRNA. This is sufficient for 10 to 60 assays with Research Genetics or Facility arrays. High-density cDNA arrays on glass use 1ug and Affymetrix arrays use 10ug aRNA. Speedvac sample to concentrate if necessary. 

Second Round of Amplification:
We have successfully used this procedure to amplify message from as little as 10ng of total RNA and we are presently working to push this amount even lower. In this case it is necessary to do a second round of amplification. In our hands, we find no significant differences in the gene expression profiles detected using aRNA derived from 1,2 or 3 rounds of amplification.
To repeat the first strand cDNA synthesis, mix 1st round aRNA (0.5-1ug; if not possible to quantitated we use 1/2 the total aRNA) with DEPC water to 9ul. Add 1ul (2ug/ul) Random Hexamers and incubate at 65C for 10 minutes. Cool samples on ice. Add the following to each sample:
5uL 5X First Strand buffer 
2uL 0.1M DTT 
2uL 10mM dNTPs 
1uL RNasin 
2uL DEPC water 
1uL 0.1ug/uL Linear Acrylamide (Ambion 9520, 5mg/mL) 
2uL Superscript II reverse transcriptase 
Incubate sample for 1.5hrs at 42C.
To denature aRNA:cDNA hybrid, heat sample for 3 minutes at 95C, then place on ice.
For second round of double-strand cDNA synthesis, add the following to each sample:
100uL DEPC water 
15uL 10X second strand buffer 
3uL 10mM dNTPs 
1uL E. coli DNA ligase 
4uL E. coli DNA polymerase I 
1uL RNaseH 
1uL (1ug/ul) oligo-dT/T7 primer
Incubate for 2 hours at 16C. Stop reaction with 10ul 0.5M EDTA and incubate for 10 minutes at 65C.
Repeat cDNA cleanup and T7 in vitro transcription as described during the first round of amplification (section 4)
The second round amplification is approximately 10-100 fold. First strand/second strand synthesis may be "visualized"/quantitated by incorporating 32P if necessary. See Molecular Cloning (Maniatus).
Modified Eberwine protocol can be accessed at http://cmgm.stanford.edu/pbrown/protocols off the main web site: http://brownlab.stanford.edu/protocols.html.
Van Gelder, R.N., von Zastrow, M.E., Yool, A., Dement, W.C., Barchas, J.D., and Eberwine, J.H. (1990) Amplified RNA synthesized from limited quantities of heterogeneous DNA. PNAS (87): 1663-1667.
Wang, E., Miller, L.D., Ohnmacht, G.A., Liu, E.T., and Marincola, F.M. (2000) High-fidelity mRNA amplification for gene profiling. Nat Biotechnol (18): 457-459. *second round protocol derived from this paper. 

Reagents
Eberwine T7 oligo, 1ug/ul 5'- AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T15 -3', Genosys, must be gel purified or better.
Trehalose, Sigma #T5251 25g $36.35
dNTP Mix (Pharmacia 27-2035-01), core #4125
Superscript II Reverse Transcriptase, (Gibco-BRL 18064-014) core #5159, $144.91
Linear acrylamide, 0.1ug/ul, Ambion #9520 $80 or prepare yourself
RNasin (Promega N2111) core #4175 $61.46, or #4174 $153.53
DNA Ligase, E. coli NAD, New England Biolabs #205L $176
E. coli DNA polymerase I, New England Biolabs #209L $200 
Ribonuclease H, (Gibco-BRL 18021-014), core #5141, $67
Random Primers (N)6 (Roche 1034731) core #4170, $106.29
Phase Lock Gel tubes, 0.5ml, Eppendorf #0032005.055 $105
ChromaSpin DEPC H2O -30 column, Clontech #K1331-1 $80
ChromaSpin DEPC H2O-100 column, Clontech #K1332-1 $85
Nicotine Adenine Dinucleotide, Calbiochem #481915 1g $49
T7 Megascript Kit from Ambion, #1334 $215
Absolute alcohol
Phenol:Chloroform:Isoamyl alcohol (25:24:1), Fisher BP-752-400

Solutions

1.7M trehalose, saturated stock solution prepared with DEPC H2O, store aliquots at -20
10mM dNTP mix
10X Second Strand Buffer
200mM Tris, pH 6.9 
900mM KCl 
46 mM MgCl2 
1.5mM Nicotine Adenine Dinucleotide (calbiochem 481915) 
100mM (NH4)2SO4 

7.5M ammonium acetate, prepared in DEPC H2O and 0.2 micron filter,
1M NaOH, 2mM EDTA first round cDNA stop solution 
0.5M EDTA
To "visualize" RNA (Total, mRNA, aRNA) quantity/quality:
RiboGreen Assay- 
use for quantity analysis of small yields of RNA (50-150ng): View product information and download Protocol pdf file from the following web address: http://www.probes.com/servlets/product?region=Select+Region&item=11490
SyberGreen Assay-
use for quality analysis of small yields of RNA (100-300ng/sample): for Protocol: http://www.bioproducts.com/technical/sybrgreeniandiigelstains.shtml, and Product info: http://www.bioproducts.com/products/sybrgreengelstains.asp?
Part=50512,50513,50522,50523,50530

Quick RNA gel-
This quick gel is sufficient to tell whether your RNA is OK. The upper Ribosomal band always appears more intense than the lower.
1. 1% Agarose Gel:
a) Dissolve 1.0g agarose in 90mL DEPC treated H2O by heating. (Scale volume to size of gel)
b) When agarose is cool enough (~55 C), add 10mL 10xRRB(RNA running buffer)
c) Pour gel - we usually use a mini-gel to preserve sample.
2. Bring 0.5 -1ug Total RNA to 7.5uL (~2ug for mRNA or aRNA)
a) Add 1uL 10x RRB
b) Heat 5min at 65 C, chill 5 min on ice.
c) Add 1.5uL of a 2:1 mix of RNA loading buffer and Ethidium Bromide (10ug/ul).
d) Phi X can be used as an approximate size standard, but if the DNA is highly nicked it will result in a smear when it is denatured by the RNA loading buffer
10x RNA Running Buffer (RRB)0.2M Hepes 23.9g Hepes
10mM EDTA 10mL 0.5M EDTA
39.6mM NaOAc 6.6mL 3M NaOAc
500mLs total at pH 7.0 (add 10N NaOH to adjust pH)
RNA Loading Buffer
80% (v/v) deionized Formamide
1mM EDTA, pH 8.0
0.1% bromphenol blue
0.1% xylene cyanol
heat samples to 90C 1 minute before loading on gel
Run Gel 100-115 V for approximately 2.5X3.5cm gel. Just don't let it boil
Amplified RNA vs Total RNA on a 1% Agarose Gel

Lanes 1-9 
200ng, as measured by OD (260nm/280nm), of 9 different amplified Human RNA (amplified from 0.5-1ug Total RNA) samples from patients were loaded into Lanes 1-9. The OD reading for the sample in lane 9 was either off or the sample was not completely resuspended when the OD was measured.
Lane 10 
1uL of a 200bp molecular weight marker from PGC (Gene Choice DNA Ladder I) was loaded into lane 10.
Lanes 11-17 
2ug of 7 different Human Total RNA samples from a cell line were loaded into lanes 11-17. (back to top)