3.3.1. Preparing Sense and Antisense frq Riboprobes and Cold Controls
Here we describe the generation of RNA transcripts in vitro transcribed using a PCR amplification product as template (see also Note 11). The generation of the PCR template is described in Subheading 126.96.36.199. Riboprobes are produced when radioactively labeled nucleotides are incorporated during the transcription reaction (described in Subheading 188.8.131.52.). When no radioisotopes are present in the transcription reaction the RNA transcripts produced can be used as cold controls (described in Subheading 184.108.40.206.). The in vitro transcription protocol used is essentially as described in the MAXIscript® kit instruction manual (Ambion) (7).
1. Perform a PCR reaction under standard conditions (16) using M13 forward and reverse primers and 25 ng pKAJ104 plasmid DNA. The vector pKAJ104 is a pBluescript-derivative that contains a 2.2kb £coRI fragment from within the N. crassa FRQ open reading reading frame (17,18).
2. Run a small amount of PCR product on a standard agarose gel (15) to verify that a single amplification product of approx 2.5 kb has been produced. This PCR product (PCR104) contains the N. crassa frq sequences flanked by T3 and T7 promoter sequences.
3. Clean up the remainder of the PCR product through a QIAquick PCR spin column (Qiagen).
4. Estimate the DNA concentration by spectrophotometric analysis or agarose gel electrophoresis (15).
In vitro transcription:
1. Thaw MAXIscript reagents on ice and briefly vortex the 10X transcription buffer.
2. Add at room temperature the following reagents in the given order to two 1.5-mL Eppendorf tubes — one tube for the reaction to produce sense RNA transcripts (using T3 RNA polymerase), the other to produce antisense RNA transcripts (using T7 RNA polymerase). Take appropriate precautions when working with 32P-labeled radioisotopes. Mix the solutions by slowly pipetting up and down when adding the last two reagents.
4. Add 1 pL of RNase-free DNase I (2 U/pL), mix with the pipet tip, and incubate for a further 15 min at 37°C, to degrade the template DNA.
Removal of free nucleotides:
5. Resuspend the contents of a Micro Bio-Spin P-30 chromatography column (BioRad). Remove air bubbles by flicking the tube. Snap off the tip of the column and place column in a 2.0-mL Eppendorf tube. Take off the top cap and centrifuge at 1000g for 2 min.
6. Place column in a fresh 1.5-mL Eppendorf tube.
PCR104 (~0.5 pg) 10X transcription buffer in 8.5 pL 2 pL
1 pL each
[a-32P]UTP (800 Ci/mmol) T3 or T7 RNA polymerase
7. Add 50 ^L of RNAse-free H2O to the 21 ^L reaction volume from step 4 and carefully pipet the sample onto the center of the column.
9. The solution collected in the tube is the cleaned RNA probe, ready for use in Northern hybridization (see Subheading 3.3.3.). Transfer 1 ^L of riboprobe into a fresh tube and count the incorporation in a scintillation counter (cpm/^L) by means of Cerenkov counting.
To produce sense and antisense frq cold controls, in vitro transcription reactions are performed, essentially as described for riboprobes (see Subheading 220.127.116.11.), using nonlabeled NTPs only.
1. Add at room temperature the following reagents:
10X transcription buffer 5 ^L
T3 or T7 RNA polymerase 5 jxL
2. Incubate reaction for 1 h at 37°C.
3. Add 2 ^L of RNase-free DNase I (2 U/^L), mix with the pipet tip, and incubate for a further 15 min at 37°C.
4. To remove free nucleotides clean up through a QIAquick PCR spin column (Qiagen) or an RNeasy spin column (Qiagen).
5. Estimate the RNA concentration by spectrophotomeric analysis or gel electrophoresis.
6. Using RNase-free H2O, make serial dilutions to obtain RNA concentrations ranging from 1 to 100 pg/^L. Store at -80°C.
To be able to normalize the frq-specific hybridization signals to an internal standard, a DNA probe from the N. crassa 18S ribosomal RNA gene is prepared, using random primed labeling (Random Primed DNA Labeling Kit; Roche), essentially as described in ref. 19.
1. Take up 50 ng PCR product (containing a fragment of the N. crassa 18SrRNA gene) in 11.5 ^L in a screw-top Eppendorf tube.
2. Boil for 10 min to denature the template DNA, and place immediately on ice.
3. Briefly spin down to collect all liquid to the bottom of the tube and replace on ice.
4. Add the following reagents at room temperature:
Reaction mix (containing hexanucleotides mix) 2 ^L
10 mM dATP/dGTP/dTTP 1 ^L each
Klenow fragment DNA polymerase I 1 ^L
5. Incubate the reaction for 3 to 4 h at 37°C.
6. Stop the labeling reaction by adding 2 ^L 0.2 MEDTA and incubating for 10 min at 65°C.
7. Increase the volume by adding 50 ^L H2O and remove nonincorporated isotope by cleaning the probe using a Micro Bio-Spin P-30 chromatography column (BioRad) as described under Subheading 18.104.22.168.
8. Transfer 1 ^L of cleaned-up DNA probe into a tube and count the incorporation of label in a scintillation counter (cpm/^L). The remainder of the DNA probe is ready for use in Northern hybridization (see Subheading 3.3.3.) or can be stored at -20°C for up to 2 wk.
3.3.3. Prehybridization, Hybridization, and Washes
22.214.171.124. Using frq Sense and Antisense Riboprobes (see Note 13)
1. Preheat hybridization oven and Northern hybridization buffer to 65°C.
2. Prehybridize two blots separately in two large hybridization bottles for 1 to 2 h at 65°C in 10 mL Northern hybridization buffer.
3. For each riboprobe calculate the amount of buffer needed to obtain a probe concentration of 2 x 106 cpm per mL hybridization buffer (see Note 14).
4. Accordingly, adjust volumes in each hybridization bottle to between 5 and 15 mL.
5. Add approx 900 ^L Northern hybridization buffer (65°C) to approx 70 ^L of each riboprobe (as obtained in Subheading 126.96.36.199., step 9).
6. Carefully pipet riboprobes into the buffer within the hybridization bottles (see Note 15) and gently swirl to mix.
8. After hybridization remove probe-containing buffer from blot (see Note 16).
9. Firstly, rinse blots in 2X SSC, 0.1% SDS to remove excess probe, then wash five times for 20 to 30 min in approx 100 mL liquid each, as follows:
a. Two washes using 2X SSC, 0.1% SDS at room temperature.
10. Take blots out of the hybridization bottles and wrap in cling film while still damp (see Note 17).
11. Expose RNA side of blots to a Phospholmager screen (Bio-Rad) or X-ray film for 2 h to overnight.
After Northern hybridization signals (using sense and antisense frq riboprobes) have been scanned, the blots can be reused immediately for hybridization with the 18S ribosomal RNA probe (see Note 18). Alternatively, Northern blots can be stored in the dark at room temperature for many months prior to ribosomal hybridization. Northern hybridization using a randomly labeled DNA probe is essentially as described above for hybridization with riboprobes (see Subheading 188.8.131.52.). Minor differences are stated below.
1. Use Denhardt's hybridization buffer for prehybridization and hybridization.
2. Boil the probe in a small volume of hybridization buffer (e.g., 500 ^L) prior to use, to make probe single-stranded.
Fig 2. Sense and antisense frq Northern hybridization signals. Riboprobes in vitro transcribed (using T7/T3) from a PCR product of the 2.2 kb £coRI fragment from within the FRQ ORF were used to assay the expression of Neurospora crassa sense and antisense frq transcripts, respectively. First published in ref. 14. (A) Example of circadian expression of sense frq mRNA transcripts (S frq) and antisense frq transcripts (AS frq) in constant darkness (DD) and after a light pulse. RNA used was extracted from mycelial samples harvested in the dark every 4 h for 48 h and 30 min after a 2-min exposure to saturating light. Also refer to Chapter 19. Levels of riboso-mal RNA (rRNA), visualized after hybridization with a N. crassa 18S rRNA probe, are used for normalization. (B) Example of cold controls routinely included on Northern gels to verify the integrity and sensitivity of the sense and antisense riboprobes.
3. Prehybridization, hybridization, and final washes are performed at 60°C.
4. Good results can be obtained using a probe concentration between 0.2 x 106 and 2 x 106 cpm per mL hybridization buffer (see Note 18).
5. Hybridization buffer (containing probe) can be reused up to three or four times without significant loss of signal intensities. There is no need to boil the probe before use in this case. If preferred, used probe can be stored at -20°C for reuse within 1 to 2 wk.
The hybridization signals can be visualized by developing the X-ray film or by scanning the Phospholmaging screen using the software package QuantityOne (BioRad). Examples of Northern hybridization results can be found in Fig. 2. Using QuantityOne the intensity of individual hybridization signals can be quantified. As the sense/antisense frq signal and the 18S riboso-mal RNA signal are both coming from the same RNA sample on the same blot, the levels of ribosomal RNA can be used to normalize the N. crassa frq-spe-cific signals.
1. Always use RNase-free glass- and plasticware. Wear gloves at all times. Single-use plasticware, such as pipets and Falcon tubes, are recommended. Plastics for multiple use should be soaked in 0.5 M NaOH for at least 30 to 60 min (no adverse effects are observed when soaking for longer periods, such as hours or overnight) and rinsed thoroughly with RNase-free MilliQ H2O before use. Bake glassware for at least 4 h at 200°C. There is no need to bake glassware after each use; just rinse with RNase-free MilliQ H2O (see also Note 2). If time constraints demand, glass- and plasticware can also sprayed and wiped with RNase-ZAP (Ambion) and rinsed with MilliQ H2O.
2. In our hands there was no need to use DEPC-treated water (15). Under different lab conditions the use of DEPC may be necessary. RNA was dissolved in RNase-free H2O (Qiagen) or MilliQ H2O, which had been autoclaved and stored in Duran bottles that were used solely for that purpose. For all buffers, MilliQ H2O was used and autoclaved as stated. There is no need to autoclave the gel running buffer, blotting buffers or buffers used for post-hybridization washes.
3. There is no need to autoclave 10X MOPS, but, if preferred, the buffer may be autoclaved. Autoclaving and exposure to light yellows the buffer. Straw-colored buffer can be used without any problems. When yellow color darkens, do not use. There is no need to autoclave 1X MOPS gel running buffer.
4. To optimize the transfer of RNA during the blotting process, use low-percentage agarose gels (<1.3%), a diluted EtBr stock (as EtBr can adversely affect the blotting efficiency of RNA), and, if possible, avoid UV exposure of RNA prior to blotting (i.e., do not take gel pictures, as RNA may crosslink to the gel, reducing the transfer efficiency).
5. It is recommended to prepare two gels of identical thickness to allow the subsequent, separate blotting process (see Subheading 3.2.) to be as similar as possible. This can be easily established by using clean 50-mL Falcon tubes or a baked 100-mL measuring cylinder to aliquot 98 mL (allowing for evaporation and inaccuracies) into each gel cast.
6. If preferred, gels can be transferred into the 1X MOPS running buffer in the electrophoresis gel tank within the hour. Take care when handling, as formaldehyde agarose gels are more fragile than same percentage TBE/TAE agarose gels.
7. To prevent confusion at later stages make the gels different by varying the positions for empty wells and/or cold controls. Blots from the gels will be probed with sense and antisense riboprobes from the same gene, so keep the sequence of the samples identical for easier and more visual-friendly comparisons. Make sure the highest concentration of cold control is always loaded away from the samples, and never loaded into a well adjacent to sample RNA. If possible, leave one or several empty wells between samples and cold controls.
8. In our experience overnight runs work best. Do not run faster than 40 V. To obtain reproducible results without taking a picture of the Northern gels (see Note 4) we successfully used "480-500 Volthours" as rule of thumb — e.g., 16 h at 30 V equals 12 h at 40 V and 24 h at 20 V.
9. As support either the gel casting tray turned upside down or a glass plate supported on four caps from 50-mL Falcon tubes can be used.
10. The time needed for complete transfer of RNA depends on the size of the transcript of interest and on the percentage of agarose used. Shorter periods are mentioned (4.5 to 6 h ), but overnight transfer is convenient.
11. It is also possible to use plasmid DNA as template for the in vitro transcription reaction. To prevent run-on transcription of vector, DNA should be cut with an appropriate restriction enzyme (3' overhangs should be avoided ). However, as one can never be sure of a full 100% digestion, it is safer to use a PCR template, in order to obtain "pure" sense and antisense riboprobes.
12. The in vitro transcription reaction can be optimized by adding "cold" UTP to the reaction mix, resulting in more full-length transcripts. Although not always essential for a good hybridization result, the use of a full-length probe generally results in less background (7). The amount of cold UTP to be added should be tested empirically. This can be achieved by running 1 ^L diluted riboprobe (~2 x 104 cpm), from samples that contained increasing amount of cold UTP, on a 4% denaturing polyacrylamide gel (15). In our hands, a final concentration of 10 to 20 ^M cold UTP gave satisfying results for the sense and antisense frq riboprobes described here.
13. At the hybridization stage it is critical to conscientiously note which blot will be exposed to which riboprobe (sense or antisense). Write it down. Remember that each riboprobe may give a similar (if not identical) hybridization result.
14. Lower probe concentrations, as low as 1 x 106 cpm per mL hybridization buffer, may give satisfying results. However, as antisense frq levels are very low in the dark (14), this may not be optimal.
15. Do not pipet the approx 1 mL of riboprobe directly onto the blot, as exposure to very high concentration of labeled probe may cause unequal hybridization results.
16. Reuse of riboprobes is possible once or twice within 3 to 4 d. The riboprobe can be used directly on another prehybridized blot or can be stored at -20°C. Incorporated radioactive nucleotides will degrade the RNA probe, resulting in reduced sensitity/poor results after several days.
17. Double-wrapping of blots is recommended to prolong lifespan.
18. As ribosomal messages are abundant the hybridization signal obtained with a ribosomal probe is strong. Therefore, there is no need for blot stripping or waiting for the decay of frq hybridization background signal at the position of the ribosomal band. Also, lower probe concentrations, as low as 2 x 105 cpm per mL hybridization buffer, give rise to reliable and good quantifiable Northern hybridization signals.
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