3.10.1 Extraction of RNA from yeast and reaction mixtures
Cells or mixtures were treated as indicated before RNA preparation. Extraction of total RNA from yeast pellets or liquid mixtures (see nucleo-cytoplasmic fractionation) as well as cytoplasmic RNA was performed using the NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s instructions. RNA that was obtained by RNA- Co-IP experiments (see 3.13.2) was purified using Trizol®. 1 ml Trizol® was added to the beads with the bound protein-RNA complexes and incubated with agitation at 65 °C for 10 min. Addition of 200 µl chloroform lead to formation of two phases – one aqueous, one organic, which were separated by centrifugation at 20,000 g for 10 min. The resulting upper phase was mixed with 1 µl glycogen and 500 µl isopropanol to precipitate the RNA. After centrifugation for 30 min at 20,000 g, 4 °C the pellet was washed once with 70 % ice-cold ethanol and let dry. RNA was resuspended in DEPC-treated deionized water, concentration measured and kept at –20 °C for short and at -80 °C for long term storage.
3.10.2 Quantitative RT-PCR (qRT-PCR)
Quantitative RT-PCR was used to examine relative induction of certain genes or the differences in protein-bound RNA quantities. This method is based on molecules like SYBR® Green that become fluorescent upon binding to double stranded DNA. After each PCR- cycle the
48 fluorescence is measured and thus the DNA product increase over time can be correlated with the starting amount of DNA. Equal amounts of RNA were transcribed into coding DNA (cDNA) using the Maxima reverse transcriptase and following manufacturer’s instructions. In general, random hexamer primer were used. As negative controls (RT-) samples without reverse transcriptase were treated equally. The resulting cDNA was diluted 1:20-1:50 depending on the amount of transcribed RNA. For analysis of ribosomal RNAs dilutions again were further diluted 1:1,000 because of the high abundance of these RNAs. 5 µl of diluted cDNA was mixed with 2x qRT-PCR master mix and gene specific primers (final concentration 0,08 µM) in 15 µl.
Reactions and analysis were carried out using mainly the qPCR Cycler CFX Connect (BioRad) and the following program:
PCR program
95 °C 5 min Initial denaturation 95 °C 5 s Denaturation
60 °C 10 s Annealing 45 cycles
60 °C 10 s Extension 65 °C–95 °C Melt Curve
At the end of the PCR a melt curve was recorded to verify that only one specific product was produced. Resulting Ct - values were normalized where applicable and/or fold changes were set in relation to total RNA, a wildtype isolate or a specific gene according to requirements of the experiment and basically applying the 2-ΔΔCt method (Livak and Schmittgen, 2001).
3.10.3 Synthesis of digoxigenin labeled RNA probes and oligonucleotide tailing
Digoxigenin (DIG)-labeled probes of 200-300 nt were used to visualize RNA in Northern Blot approaches or in situ hybridization microscopy studies. Templates for RNA specific probes were prepared by PCR using primers pairs with one carrying a T7-promoter (HK 2022 and HK 2023 for the GFP probe). These purified templates were used for in vitro run off-transcription reactions with DIG-UTP labeling mix (Roche). This mix contains all four NTPs that naturally occur in RNAs together with DIG-11-UTP in smaller amounts, which leads to incorporation of DIG-UTP every 20th-25th nucleotide depending on RNA composition.
Reactions were prepared as suggested by the manufacturer.
Materials and Methods
49 DIG-labeling reaction 20 µl 200-250 ng DNA template
1x Transcription buffer 1x DIG RNA labeling mix 20 U RiboLock RNase Inhibitor 40 U T7 RNA polymerase
After incubation at 37 °C for 2 h the DNA template was digested with 2 µl DNase I for 15 min at 37 °C. RNA probes were precipitated with 0.25 volumes 4 M LiCl, 1 µl glycogen and 3 volumes 100 % ethanol overnight at -20 °C. The following centrifugation at 20,000 g for 30 min pelleted the RNA, which was washed with 70 % ethanol and air-dried. The probe was resuspended in 20 µl formamide, 20 µl 0.5x TE pH 7.5 and 60 µl HybMix before stored at -20 °C.
Hybridization mix (HybMix) 50 % (v/v) deionized formamide 5x SSC
1x Denharts solution 0.1 mg/ml Heparin 50x Denharts solution 1 % (w/v) Ficoll
1 % (w/v) Polyvinylpyrrolidone 1 % (w/v) BSA
For identification of poly(A)-containing RNAs on Northern dot blots, a DIG-labeled oligo-dT probe was prepared. Since in vitro transcription of poly(A) stretches is not very effective a different method was chosen. A 50 nt long dT oligo was 3’ end tailed with approximately 100 nt DIG-dUTP and dATP using an DIG-dUTP tailing kit (Roche) and following the enclosed protocol. The probe was precipitated and restored as described above.
3.10.4 Nucleo-cytoplasmic fractionation
Extraction of cytoplasm was, with modifications, performed as described (Sklenar and Parthun, 2004). For nucleo-cytoplasmic fractionation cells were grown to log-phase in selective medium.
After harvest cells were washed once with 1ml YPD/ 1 M Sorbitol/ 2 mM DTT and resuspended in YPD/ 1 M Sorbitol/ 1 mM DTT. Cells were spheroblasted using Zymolyase and diluted in 50 ml YPD/ 1 M Sorbitol to recover for 30 min at 25 °C and shifted to indicated temperature if applicable. Cells were put on ice, centrifuged at 900 g for 5 min and resuspended in 500 µl Ficoll buffer.
50 Ficoll buffer pH 6.0 18 % (w/v) Ficoll 400
10 mM HEPES
Buffer A pH6.0 50 mM NaCl
1 mM MgCl2 10 mM HEPES
Total RNA sample was taken and subsequently cells were broken by addition of 1 ml buffer A.
The suspension was mixed and centrifuged at 1,500 g for 15 min. The supernatant was used for cytoplasmic analyses. To verify correct fractionation of the cytoplasmic lysates, samples were analyzed in western blot for the presence of the cytoplasmic protein Zwf1 and nucleolar protein Nop1. RNA was purified as described above
3.10.5 RNA agarose gels, Northern blot and Dot blot experiments
Total or cytoplasmic RNA was analyzed in Northern blots and analysis was performed as described earlier (Sambrook et al., 1989; Wu et al., 2014). For gel-electrophoresis all solid material was cleaned with 0.1 M NaOH and all aqueous solutions treated with 0.1 % DEPC to avoid RNA degradation by contaminating RNases.
10x MOPS pH 7.0 200 mM MOPS
50 mM Sodium acetate 10 mM EDTA
RNA loading dye 50 % (v/v) deionized formamide 6 % (v/v) formaldehyde
1x MOPS
25 ng/ml ethidium bromide 10 % Glycerol
Bromophenol blue and Xylene cyanol
20x SSC 3 M NaCl
300 mM Sodium Citrate
Gels containing 1x MOPS, 1 % agarose and 2 % formaldehyde were cast under the hood. Equal amounts of RNA were mixed 1:2 with RNA loading dye, denatured for 10 min at 65 °C and chilled on ice. Samples were loaded onto the gel and let run in 1x MOPS at 120 V for 1-2 h.
Integrity of the RNA and equal loading was analyzed under UV light before the gel was washed once with DEPC-treated water and twice with 20x SSC to remove the formaldehyde before blotting. RNA was transferred to a nylon membrane with dry capillary blot overnight. For this the gel was set on a glass plate followed by the membrane, 3 layers dry Whatman paper and a
Materials and Methods
51 stack of paper towels, 500 g weight compressed the setup. The next day, the transferred RNA was cross-linked to the membrane using 5,000 J/cm2 UV light for 7 min and baking at 80 °C for 2 h. For dot blot experiments the eluted RNA was spotted on the membrane, let dry, cross-linked as above and treated the same.
Hybridization buffer pH 7.2 500 mM Sodium phosphate pH 7.2 7 % (w/v) SDS
1 mM EDTA
1M Sodium phosphate pH 7.2 68.4 ml 1M Na2HPO4
31.6 ml 1M NaH2PO4
5x Maleic acid buffer pH 7.5 0.5 M Maleic acid 750 mM NaCl
1x Blocking solution 1 % Blocking Reagent (Roche) 1x Maleic acid
Washing buffer 1x Maleic acid
0.3 % (v/v) Tween 20 Detection buffer pH 9.5 100 mM Tris pH 9.5
100 mM NaCl
The membrane was prehybridized with hybridization buffer for 1 hour at hybridization temperature (42 °C for oligo dT probes, 68 °C for gene specific probes) with agitation. 1 µl of the respective probe was added to the membrane and hybridized overnight. Next day the membrane was washed once in 2x SSC, 0.1 % SDS and 1x SSC, 0.1 % SDS at room temperature und twice with 0.5x SSC, 0.1 % at hybridization temperature for 15 min. After shortly washing with washing buffer, the membrane was blocked for 30 min in 1x blocking solution and incubated with anti-DIG antibody (1:10,000 in blocking buffer) for 60 min. Two 15 min washing steps with washing buffer were performed before the membrane was equilibrated in detection buffer and CSPD (diluted 1:100 in detection buffer) was applied. To reduce background, the membrane was incubated at 37 °C for 10 min before detection of the signal with either the Fusion camera or X-ray sensitive films (Fuji) depending on signal intensity.
52