3. METHODS
3.2. RNA-Techniques
Methods
Table 3-16 Reverse Transcription Reaction for Stem-loop Primers
Reagents Amount
First strand buffer [5x] 4 μl
DTT [0.1 M] 2 μl
RNAseOut [40 U/μl] 0.5 μl
Superscript III RT [1 U/μl] 0.25 μl
DEPC-H2O to 19 μl
Subsequently 1 μl RNA was added and a pulsed RT reaction was performed, shown in table 3-17.
Table 3-17 Reverse Transcription Program for Stem-loop Primers
Temperature Time Cycles
16 °C 30 min 1
30 °C 30 s
42 °C 30 s
50 °C 1 s
60
4 °C ∞
After cDNA synthesis 0.4 μl RNAse H [200 units/μl] was added and the mixture was incubated at 37
°C for 20 min. RNAse H was inactivated by heating at 65 °C for 10 min and tubes were put on ice prior to use or stored in aliquots at -20 °C.
RNA was subsequently transferred to a Nitran Nylon membrane with the TurboBlotter System. The transfer was performed in 2x SSC (Sodium chloride, Sodium Citrate)/0.5 M NaOH for 3 h.
Hybridization of the membrane with oligonucleotide probes was done in a similar fashion to the small RNA Northern Blot (see 3.2.2.1). Probes < 100 bp synthesized by PCR were labeled with the DNA Labeling System and the blot was hybridized in UltraHyb hybridization buffer according to the manufacturer’s instructions. The blots were developed for 1-3 days at -80 °C and the radioactivity was detected with X-ray films
Table 3-18 10x MOPS Buffer
Reagents Amount
MOPS 200 mM
Sodium Acetate 50 mM
EDTA 10 mM
NaOH to pH 7
Table 3-19 Loading Dye for Northern Blot Samples
Reagents Amount
saturated aqueous bromophenol blue solution 4 μl EDTA [500 mM], pH 8 20 μl Formaldehyde [37%] 180 μl Glycerol [100%] 500 μl
Formamide 771 μl
10x MOPS 1 ml
DEPC-H2O to 2,5 ml
3.2.2.1. Small RNA Northern Blot
The detection of small RNAs such as miRNAs was done by separating 14 μg total RNA on a 15%
8 M urea SDS gel. Components of the gel are listed in table 3-20. First urea was dissolved in buffer, acrylamide solution and buffer by warming the mixture to 37 °C and stirring. Then APS and TEMED were added and the gel was casted between to glass plates for polymerization.
Table 3-20 8 M Urea Gel for Small RNA Northern Blot
Reagents Amount
Urea 12 g
10x TBE 2.5 ml
30% Acrylamid/Bisacrylamid (ratio 19/1) 12.5 ml
DEPC-H20 6.7 ml
APS 160 μl
TEMED 16 μl
The gel was pre run for 1 h at 45 mA. Samples were loaded after mixing 15 μl RNA with 15 μl formamide containing loading dye and heating the mixture for 2 min at 70 °C. RNA was then separated for 45 min at 35 mA. Afterwards the gel was stained in a 4% ethidium bromide solution and bands were evaluated for integrity by UV light. After washing the gel in 1x TBE (Tris, Borate,
Methods
1x TBE for 2 h at 2.26 mA/cm2. The membrane was washed in 1x TBE, dried and cross-linked with UV light. Pre-hybridization was performed for 1 h at 37 °C in hybridization buffer in a rotating hybridization oven. At the same time the oligonucleotide probe was labeled for 1 h at 37 °C in a water bath.
Table 3-21 Labeling Reaction for Small Oligonucleotide Probes
Reagents Amount
DEPC-H2O 16 μl
10x PNK buffer 5 μl Probe [10 mM] 2 μl
PNK [6 U/μl] 2 μl
P32-dATP [mCi] 25 μl
The PNK activity was inactivated by heating the probes for 10 min at 65 °C. Nucleotides which were not incorporated were separated from the probe using G-25 Sephadex columns according to the manufacturer’s instructions.
The hybridization buffer was changed and the labeled probes were added. Membranes were then incubated over night at 37 °C under constant rotation. The next day membranes were washed twice in pre-heated wash buffer (2x SSC, 0.1% SDS), once at 37 °C for 1 min, once for 30 min at RT and twice for 30 min in RT wash buffer at RT. Membranes were then exposed to X-ray films or imaging plates for 1-3 d at -80 °C or at RT, respectively. X-ray films were developed on a E.O.S. processor and image plates were analyzed on the BAS-Reader. The evaluation was performed with the AIDA Software.
For multiple hybridization of the blot with different probes, the previously bound probes were removed by incubating the membrane in 0.5% SDS for 2 h at 60 °C in a rotator.
Table 3-22 TBE Buffer
Reagents Concentration
TRIS base 89 mM
Boric Acid 89 mM
EDTA 20 mM, pH 8
Table 3-23 Formamide Loading Dye
Reagents Concentration
Formamid 95% (v/v)
Bromphenol blue 0.09% (w/v) Xylene cyanol FF 0.09% (w/v)
Table 3-24 20x SSC Buffer (Sodium Chloride, Sodium Citrate)
Reagents Concentration
NaCl 3 M
Sodium citrate 0.3 M
HCl to pH 7
Table 3-25 Northern Blot Wash Buffer
Reagents Concentration
SSC 2x SDS 0.1%
3.2.3. Cloning of Small RNAs
A cloning strategy for small RNAs was first described by Pfeffer et al. and was used for the identification of diverse small RNA species. In this work new miRNAs were predicted with the VMIR Programm and verified by small RNA Northern Blotting. To determine the exact 5’-ends of the confirmed miRNAs a modified protocol from Pfeffer (Pfeffer et al., 2005a) was used.
Total RNA (500 µg) from positive cell lines or transfected cells were separated on a 15%
polyacrylamide gel containing 8 M Urea (see 3.2.2.1) and stained with ethidium bromide. RNA in the range of 18-26 nt was excised, crushed into small pieces in a 2 ml low bind Eppendorf tube and eluted rotating over night at 4 °C with 2-3 volumes of 0.3 M NaCl-solution. The next day gel pieces were centrifuged (2000 rpm, 4 °C, 5 min, Centrifuge 5417 R, Eppendorf) and the supernatant was transferred in a new tube. RNA was precipitated in the presence of 1 μl GlycoBlue or glycogen with 3-4 vol of 100% EtOH at -20 °C over night. The RNA was pelleted (16000 g, 4 °C, 10 min), dried in the SpeedVac and resolved in 30 μl DEPC-H2O. For dephosphorylation, the components listed in table 3-26 were mixed and incubated at 50 °C for 30 min.
Table 3-26 Dephosphorylation Reaction Mixture for Small RNA Cloning
Reagents Amount
RNA 27 μl
NEB buffer 3 1 μl
RNAseOut [40 U/μl] 1 μl
CIP [1 U/μl] 1 μl
RNA was then extracted with phenol-chloroform (see 3.1.9) and resolved in 11.5 μl DEPC-H2O.
Ligation of the 3’-linker was carried out by adding 1 μl of linkers [100 μM] to 10 μl RNA. The mixture was heated to 65 °C for 3 min. Then components listed in table 3-27 were added.
Table 3-27 Linker Ligation Reaction Mixture for Small RNA Cloning
Reagents Amount
ATP [10 mM] 2 µl
RNase Out [40 U/µl] 1 µl 10x RNA-Ligase Buffer 2 µl
BSA 2 µl
T4-RNA-Ligase 1 µl
The ligation was performed at 16 °C over night. To get rid of unbound 3’-linker, the ligated RNA was separated on a 8.5 cm 15% polyacrylamide gel containing 8 M Urea. RNA in the range of 52-58 nt
Methods
ligation the isolated RNAs have to be phosphorylated at their 5’end. Therefore, reagents listed in table 3-28 were added to 10 µl RNA and the mixture was incubated at 37 °C for 30 min.
Table 3-28 Phosphorylation Reaction Mixture for Small RNA Cloning
Reagents Amount
10x PNK buffer 2 µl ATP [100 mM] 0.4 µl
PNK 0.5 µl
DEPC H2O 7.1 µl
Following this, 37 µl DEPC-H2O and 3 µl 5 M NaCl solution were added and RNA was extracted with PCI (see 3.1.9). Ligation of the 5’-linker was done in the same way as the ligation of the 3’-linker with subsequent purification on a 8.5 cm 15% polyacrylamide gel containing 8 M Urea. RNA in the range of 85-91 nt was cut out and isolated as described above. The RNA was then reverse transcribed to cDNA using the 3’-outer primer (see also 3.1.11.3). For unspecific amplification of the ligated small RNAs a PCR was carried out with the 5’- and 3’-outer primers (3.1.10). If the yield was too low a second PCR was performed by using the 5’- and 3’-inner primers. To sequene the 5’-ends of single cloned small RNAs, products were PCR amplified with a specific 3’-primer for each identified miRNA and the 5’-outer or -inner primer. Products were purified on 2% agarose gels, extracted, cloned into a TA-cloning vector (3.1.6.1) and then sequenced (3.1.8).