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The focus of this article is to optimize the protocol for in situ detection and sequencing of individual mRNA transcripts at a single cell level using padlock GAP probes. In addition to the advantages of the normal padlock probe, padlock GAP probes provide us with the ability to sequence short regions on the mRNA transcripts directly and to study multiple SNPs that are present adjacent to each other. GAP-fill polymerization is a critical step involved in circularization of the padlock GAP probes enabling downstream Rolling Circle Amplification (RCA) and Rolling Circle Product (RCP) detection. Some of the key factors that are important for GAP-fill polymerization have been optimized in this study, such as the dNTP concentration and the different kinds of DNA polymerases required for GAP polymerization. Results indicate that, using different polymerases such as Stoffel fragment and Taq Polymerase do not significantly affect GAP-fill polymerization. However, changing the dNTP concentration to 0.25mM has been found to increase the efficiency of GAP-fill polymerization. This technique has further implications in detecting biomarkers in cancer stem cells, in developmental biology and other areas to study single cell gene expression and genotyping profiles.
As the name suggests, In situ single molecule detection provides the opportunity to investigate single DNA or RNA molecule in tissues or in cells. The present molecular methods used for analysis of molecules such as DNA and RNA is conducted in a heterogeneous environment due to the limitations in isolating a single cell. Thus the methods measure the average of molecules present in a given sample. For example, consider the quantitative gene expression studies, they use the mRNA from the entire tissue or from a total cell population to study their respective gene transcripts. It has been shown that cell-to-cell gene expression variation occurs even in a synchronized cell cultures . Thus the quantitative gene expression data may not provide specific expression profile of a subset of cell population. This demands a molecular method, which enables the study of a single gene transcript in a single cell basis. In situ single molecule detection technique might prove to be vital for biomarkers analysis in clinical applications such as cancer prognosis and for studying cancer stem cells, which are hard to detect due to the heterogeneous tumor microenvironment.
In situ hybridization (ISH) is a widely used technique for detecting specific nucleic acid molecules. Different types of labeling for probes were used right from radioactive materials to antigen labeling and fluorescence labeling. Fluorescence in situ hybridization (FISH) is commonly used techniques to detect single mRNA molecule using probes labeled with multiple fluorophores  or with multiple probes labeled with single fluorophore . Using this technique multiple transcripts can be simultaneously detected from a single sample. FISH efficiency depends on the length of the probes used. As the length of the probe decreases in order to study the shorter targets; its efficiency also reduces drastically [4, 5]. Further limitation is the inability to detect targets, which are highly similar, thus the method cannot be used for allelic silencing, splice variations and to distinguish the targets of same family members .
In our study we discuss about the in situ detection of single mRNA transcripts. Any molecular method, which studies biomolecules, should consider both sensitivity and specificity. For this purpose, padlock probes have been used. Padlock probes (70-100nt) are oligonucleotides, which are linear and can be circularized after hybridizing with the target nucleic acid molecule. Padlock probe technology is based on OLA (Oligonucleotide ligation assay) . Padlock probes are circularized only if the two ends of the probe hybridize and are enzymatically ligated by a DNA ligase . Circularized probes are not formed in case of any mismatches at the ligation site of the probe hybridized to the target molecule. A single nucleotide variation in the target molecule can affect the probe hybridization. Apart from the probe's complementary sequence to the target nucleic acid molecule, it also bears a detection oligo sequence for later detection of the hybridized probe. The name "padlock" arises due to the nature of the probe, which wraps around the target nucleic acid molecule because of the helical structure of the DNA .
In situ single molecule detection method requires a very high resolution. Thus the signal that reports the presence of the target molecule should be detectable and this calls for a specific type of amplification to detect the probes. Rolling cycle amplification is an apt method for padlock probe detection. Circularized padlock probes acts as a template for the amplification . The Rolling cycle product (RCP) contains the tandem repeats of the padlock probe complementary sequence . The target nucleic acid molecule acts as the primer for this reaction. A special type of polymerase is used for this RCA. Phi29 DNA polymerase which has 3'-5' exonuclease activity uses target molecule as a primer and polymerizes linear strand of sequence complementary to hybridized padlock probe . RCP contains repeated sequences of the detection oligo sequence, which is part of the padlock probe. Using a sequence specific fluorophore, tagged oligo nucleotide can be hybridized to the RCP followed by detection of the target molecule, which is about 1um signal size . Though this method is highly standardized for DNA molecule , detection of RNA molecule using RNA as the template for ligation of the probes seems to reduce the efficiency of the detection . Sensitivity and the specificity were also compromised while using RNA as template for the probe hybridization. Recent publication by Larsson et al , has described a method to detect single mRNA transcripts in cultured cells and in frozen tissues using padlock probes targeting cDNA of the specific transcript. Complementary DNA molecule (cDNA) was synthesized after Revere transcription reaction using Lock Nucleic Acid (LNA) primer specific for the transcript. This method is highly sensitive and can identify even a single nucleotide difference in the target molecule hence, can be used for genotyping purposes.
Genotyping of the mRNA transcripts directly elucidates the functional effect of the SNPs percent in specific cells or tissue. Currently this technique has been standardized for detecting Single nucleotide variation; but to study multiple SNPs which are present in close proximity, different type of padlock probes has to be employed called padlock GAP probes. Padlock GAP probes are similar to the normal padlock probes, but once hybridized it will not ligate as the normal padlock probes, since the 3' and 5' end of the padlock GAP probes are not juxtaposed, leaving few nucleotide (4 nt) GAP between the ends. Thus, to make the padlock probe circularized, the gap has to be filled either by GAP-fill polymerization or using short nanomers probes for the GAP FILL process. Once the GAP of the padlock probe is synthesized, the RCA begins. Using a nanomer detection oligo tagged with the specific fluorophore that can be hybridized to the RCP a signal can be detected which provide us information as to which nucleotides are incorporated in the GAP and which nucleotides are complementary to the target molecule.
In this article we discuss about the optimization process of GAP-fill polymerization using padlock GAP probes. Several factors can affect the GAP-fill polymerization efficiency such as dNTP concentration, enzymes used for GAP-fill polymerization etc. The schematic representation of the procedure followed is illustrated in the figure 1.
Materials and Methods:
In our experiments we have used three different types of cell lines namely; Transformed human epithelial cells (HeLa), Human fibroblasts (BJ-hTERT) and mouse embryonic fibroblast (MEF). HeLa cells were cultured in DMEM (Dulbecco's modified Eagles medium) while MEF were cultured in DMEM without phenol red. DMEM is further supplemented with 10% FBS, non-essential amino acids, L-glutamine (2 mM), penicillin (100 Î¼g/ml) and streptomycin (100 U/ml). BJ-hTERT cells were cultured in Minimal essential media (MEM) without phenol red with the same supplements as mentioned above. All the cell lines were continuously cultured in humidified chambers (5% CO2) maintained at 37 Â°C.
Sample preparation for in situ experiments:
All the cell lines used in our experiments were seeded in culture dish containing Superfrost plus slides from Thermo scientific and left for 18-24 hrs before fixing, for cell attachment. For co-cultured experiments, MEF and BJ-hTERT cells of equal density were mixed and seeded on the dish containing Superfrost plus slides. Once the cells reach the desired confluency, they were washed once in 1XPBS and then fixed in 3% paraformaldehyde (PFA) in 1XPBS (w/v) for 30 min at room temperature. After the cell fixation process, the slides were washed twice with DEPC-PBS (1x) followed by dehydration process. Dehydration of the slides was performed by dipping the slides in ethanol, starting from 70%, 85% and 99.5%(Ethanol series) for 2 min each. Secure-seals (Grace Bio-Labs Inc) of 9 mm diameter and 0.8 mm deep were attached to the dry surface of the slides containing fixed cells. All the molecular reactions were performed inside this chamber. The maximum capacity of each chamber is 50 Î¼l of reaction volume. To improve the cDNA synthesis efficiency, 0.1 M HCL (in DEPC H20) was added to cells for 5 min at room temperature, which increase the RNA availability for the reverse transcription. After HCL treatment, cells were washed twice briefly with DEPC-PBS-Tween20 (0.05%).
LNA based incorporated cDNA primers and the padlock probes for the human Î²-actin transcript were designed based on the Genebank sequence with accession no NM_001101.3 Oligonucleotide sequences used in our experiments are summarized in Table 1.
In situ Reverse Transcription:
Samples were treated with 20 U/Î¼l of M-MuLV Reverse transcriptase (Fermentas), 0.5 mM dNTP (Fermentas), 0.2 Î¼g/Î¼l BSA (NEB), 1 Î¼M of LNA cDNA primer specific for human Î² actin (P4507), 1 U/Î¼l RNase Inhibitor (Fermentas) along with 1x concentration of M-MuLV reverse transcriptase buffer totaling 50 Î¼l of reaction volume per sample. After the addition of the RT reaction mix, slides were incubated at 37Â°C for 3 hrs. After the incubation, slides were washed twice briefly with PBS-Tween20 (0.05%). Cells were further fixed with 3% formaldehyde in PBS-Tween20 for 10 min after in situ RT reaction, which is followed by two washes with PBS-Tween20.
Padlock probe hybridization and ligation:
Padlock probe hybridization and ligation was carried out with 0.1 Î¼M concentration of each probe in a reaction mixture containing 0.75 U/Î¼l Ampligase, 1 U/Î¼l RNase inhibitor, 0.4 U/Î¼l Rnase H, 0.2 Î¼g/Î¼l BSA, 0.05 M KCL, 20% formamide along with the 1x ampligase buffer. This step is followed by incubation of slides first at 37 Â°C for 20-30 min and at 45 Â°C for 1 hour. After probe ligation, samples were washed with 2XSSC-Tween20 (0.05%) at 37 Â°C for 5 min followed by a DEPC-PBS-Tween20 wash.
Padlock GAP probe hybridization and polymerization:
Padlock GAP probe hybridization requires few more components than the normal padlock probe. In our experiments we have used a probe with a four nucleotide GAP (P4859) between the 3' and 5' end of the probe, binding to the target cDNA. GAP-fill polymerization method is used to fill the GAP to make the padlock probe a circularized molecule. Along with the components of the normal padlock probe hybridization and ligation mix, 0.1 Î¼M of GAP padlock probe is added to a reaction mix containing 0.1 U/Î¼l of Stoffel or 0.1 U/Î¼l Taq DNA polymerase (Depending on the optimization procedure explained in the Results section), 2.5 mM MgCl2 and 0.05 mM dNTPs for polymerization. dNTPs concentration may also vary between the experiments. After the padlock GAP probe hybridization, and polymerization step, slides were treated exactly the same way as the normal padlock probe samples.
Rolling circle amplification and detection of RCP:
RCA was performed using phi29 DNA polymerase that recognizes the circularized padlock probes . Ï•29 DNA polymerize (Fermentas) of 1 U/Î¼l concentration was added to a reaction mix containing 1 U/ul RNase Inhibitor, 0.25 mM dNTP, 0.2 Î¼g/Î¼l BSA, 5% glycerol long with the 1x Ï•29 DNA polymerase reaction buffer. Samples were incubated at 37 Â°C for 90 min, followed by two 1XDEPC-PBS-Tween20 washes. After the RCA, rolling circle products (RCP) were detected using hybridizing the fluorophore tagged detection oligonucleotides. Detection oligonucleotide of 0.1 Î¼M concentration was used to hybridize on the RCPs along with the 1X hybridization buffer. Hybridization took place for 15-30 min at 37 Â°C, and was further followed by a couple of washes in 1XDEPC-PBS-Tween20 buffer. Secure seals on the slides were removed and the dehydration process (Ethanol series) of the slides was performed in a similar way as mentioned before. Once the slides were dry, they were mounted with Vectashield containing 100 ng/ml DAPI to stain the nucleus of the cells. Slides were then continued for Image acquisition and analysis
Stripping and re probing of GAP fill detection probe:
To detect the RCPs of the padlock GAP probe using GAP fill probe (L11524), we stripped the RCPs, which were bound to detection oligonucleotide probe (L8748). After acquiring the images of the slides using detection oligo probe, slides were dehydrated using ethanol series as mentioned above. New secure seal was mounted back at the exact position. Samples were then washed twice with 65% formamide at 45 Â°C for 2 min. After the incubation, the samples were further washed twice with 1XDEPC-PBS-Tween20 buffer. Slides were then inspected in fluorescence microscope to look for any residual signals. Slides were again dehydrated with ethanol series. Anchor primer (L11286) was then hybridized on to the RCPs at 10 Î¼m concentration per sample, in a reaction mix containing 1x hybridizing buffer. Samples were then incubated for 20 min at room temperature. After the incubation, samples were washed twice with the washing buffer (1XDEPC-PBS-Tween20). Now GAP fill detection probe (L11524) at a concentration of 10 Î¼M per sample was used to ligate the anchor primer with a reaction mix containing T4 DNA ligase (3 U/Î¼l), ATP (10 mM) in 1X T4 DNA ligase buffer solution. Samples were then incubated at Room temperature for 30 minutes in dark. After incubation, samples were washed again with the wash buffer and dehydrated with ethanol series. Slides were then DAPI (100 ng/ml) stained using Vectashield and continued for Image acquisition and analysis.
Image acquisition and analysis
AxioPlanII epi-fluorescence microscope (Zeiss) was used to acquire the images of cultured cells after the in situ procedures. This microscope is attached with a 100 W mercury lamp, a CCD camera (C4742-95, Hamamatsu) and a computer controlled filter wheel. These filters were set to measure the excitation and the emission of DAPI and Cy3 Dye. Both 20X and 40X objectives (Zeiss) were used to acquire the images. Images were acquired using Axiovision software (Version 4.3, Zeiss). After acquiring the images, Blob Finder software (Version 3.0 Beta)  was used to digitally quantify the RCPs (Blobs) and the cells using cell nuclei stain (DAPI). Five 20X images were used to quantify the RCPs in cultured cells. Total number of RCPs per image was divided by the number of nuclei in the image to acquire the RCPs per cell count. Average of RCPs per cell count from all the five images were reported as our final RCPs per cell for each experiment.
Figure 1: Schematic representation of procedure explaining both normal and padlock GAP probe hybridization on cDNA after in situ reverse transcription reaction followed by RCP detection.
Type of oligonucleotideOligonucleotideSequencecDNA primerP4507-pLNA_ACTBhum5'-CTGACCCATGCCCACCATCACGCCC-3'Padlock probesP4190-PdACTBhum5'GCCGGCTTCGCGGGCGACGATTCCTCTATGATTACTGACCTATGCGTCTATTTAGTGGAGCCTCTTCTTTACGGCGCCGGCATGTGCAAG-3'Padlock GAP probeP4859-pdGap_ACT_4 nt
5'AGGCCGGCTTCGCGGGCGACGGCGACTATGATTACTGACTGCGTCTATTTAGTGGAGCCCTATCTTCTTTCAACGGCTCCGGCATG - 3'Detection oligoL8748-B2_DO_27+_Cy35'-TGCGTCTATTTAGTGGAGCC-3'GAP fill detection probeL11524-A
CT_4 nt_9mer comp
5' UGC AAG GCC - 3'Anchor primerL11286-ACTanchP_4 nt
5' - CAA CGG CUC CGG CAU G - 3'Table 1:
Results and Discussion:
Troubleshooting in situ cDNA detection using normal padlock probes:
Initially we started our experiments with the normal padlock probes for detecting the human Î² actin transcripts in cultured cells. Though the protocol for the normal padlock probes has been standardized , we were having some problems in acquiring good signal from RCPs. To troubleshoot this issue, we prepared fresh stock of buffers for in situ cDNA detection procedure. BJhTERT and MEF cells were co-cultured and were fixed for the in situ cDNA experiments as described in the materials and method section. Human Î²-actin transcripts were reverse transcribed using a specific LNA based primer (P4507). Although the observed signals from the RCPs was low and inefficient it was detected only in the RT+ cells which was our positive control and not in RT-cells (Negative control) (Fig. 2A) suggesting that the experiment was functioning at a lower than optimal level. This eliminated the buffer contamination possibility for the reduced detection efficiency. Next we tried using fresh batch of Rnase H enzyme, and prepared a fresh stock of phosphorylated padlock probe (P4190) specific for the human Î²-actin. In figure 2B we observed very good signal from the RCPs. HeLa cells were used for the above experiment.
Figure 2A: Detection of human Î²-actin transcripts in BJhTERT and MEF co-cultured slides.
Neg CTRL: Samples with no addition of reverse transcriptase; Human Î² actin: Î²-actin transcripts (red) detected using normal padlock probe-P4190 and DO probe L8748 Cy3 in human fibroblasts (BJhTERT). Nuclei of the cells are stained with DAPI (blue).
Figure 2B: Detection of individual Î²-actin transcripts after using Fresh batch of Rnase H and phosphorylated padlock probe P4190. Neg Ctrl: Negative control samples with no reverse transcriptase added; Human Î²-actin: Î²-actin transcripts (red) detected using padlock probe P4190 and DO probe L8748-Cy3 in HeLa Cells. Nuclei of the cells are stained with DAPI (blue). Images were taken both in 20X and 40X magnification.
dNTP concentration titration for GAP-fill polymerization:
Now that the detection of RCPs had been standardized, we continued with the optimization of in situ cDNA detection using GAP padlock probes. To start with, we decided to titrate the dNTPs concentration required for GAP polymerization. dNTPs concentration during the GAP-fill polymerization is a critical parameter for determining the efficiency of RCA and downstream detection of RCPs. As a positive control, human Î²-actin transcript is detected using padlock probe (P4190) without the GAP. Three different concentrations of dNTPs (0.25, 0.05, 0.025 mM) was used for the GAP-fill polymerization of the padlock GAP probe (P4859) with four nucleotide GAP. Stoffel fragment enzyme was added to the reaction mix. Detection oligo (L8748-Cy3) is used to detect the RCPs. As the figure 3A and B illustrates, 0.25 mM concentration of the dNTPs provide better detection efficiency of GAP padlock probes in BJhTERT cells. It was noted that the overall detection efficiency of RCP reduces by more than half while using the Padlock GAP probe compared to the normal padlock probes. Altering the dNTP concentration alone is not sufficient for improving the detection efficiency. The results indicate that, there are other major factors involved in the process which might need further optimization such as type of enzymes used for GAP-fill polymerization, concentration of enzyme, padlock GAP probe hybridization time and temperature, salt concentration and pH.
Figure 3A: Detection of Î²-actin transcripts using padlock GAP probe using different dNTP concentrations for GAP-fill polymerization. Ctrl: Detection of Î²-actin transcripts (Red) using normal padlock probe (P4190) and DO (L8748); 0.25 mM, 0.05 mM and 0.025 mM dNTP concentrations used for GAP-fill polymerizing the probe. Detection of Î²-actin transcripts using GAP padlock probe (P4859) and DO probe (L8748-Cy3). Nuclei of the cells are stained with DAPI (blue).
Figure 3B: Quantification of RCPs per cell in dNTP concentration titration experiment. 0.25 mM, 0.05 mM and 0.025 mM dNTP concentrations used for GAP-fill polymerizing the probe.
Enzyme optimization for GAP-fill polymerization:
Padlock GAP probe, needs to be polymerized to become a circularized molecule, which in turn can be amplified by RCA technique. GAP-fill Polymerization technique provides us the nucleotide sequence of the target molecule. The polymerization occurs in the padlock GAP probe after hybridization to a specific site on a target cDNA molecule and adds nucleotides complementary to the target sequence. In our experiments, we used exact matching sequence probe (GAP fill probe) which is tagged with Cy3 fluorophore to check the efficiency of the padlock GAP probe RCA and detection of RCPs as a proof of concept. In actual sequencing method, we will be using probes from a nanomer mix, in which each probe with a fixed single nucleotide position bears a specific fluorphore tag. Thus, during detection of RCPs, we can identify the probe which is bound to the RCP and providing the information of the target molecule nucleotide sequence.
Thus, enzymes efficiency to polymerize the GAP in the padlock needed to be standardized. We have tested both Stoffel Fragment and Taq DNA polymerase. BJhTERT Cells were cultured and detected for Î²-actin transcript. Fig. 4a illustrates the detected RCPs after probing with the Detection oligo sequence probe (L8748-Cy3). The same samples were then stripped (fig. 4b) to re-probe using the GAP fill probe (L11524-Cy3) (fig. 4c). Fig. 5 shows, the statistics of the RCPs per cell using different DNA polymerases compared to the control padlock probe. From the results, we can conclude that, there is a difference between Stoffel fragment and Taq while detecting using Detection oligo probe but not much of difference when GAP fill probe was used. RCP detection rate decreases tremendously, even within the Control samples, which were using normal padlock probe. Thus, irrespective of the nature of the probe we have used, the detection efficiency has reduced while using the GAP fill probe detection. This suggests that, factors such as hybridization of anchor primer to the RCP, ligation of GAP fill probe to the anchor primer and final detection reduces the overall sensitivity of the procedure.
Therefore, factors such as concentration of the anchor probe, temperature of hybridization of anchor probe to RCP, time of hybridization, salt concentration, concentration of GAP fill probe etc., need to be optimized in future. Once optimized, in situ cDNA detection and genotyping will be an efficient and cost effective method to study multiple gene transcript variations at a single cell level.
Figure 4: Detection of Î²-actin using padlock GAP probe in BJhTERT cells using Stoffel and Taq DNA polymerase for GAP-fill polymerization. Ctrl: Detection of Î²-actin transcripts (Red) using normal padlock probe (P4190). GAP Padlock probe (P4859-Î²-actin) was used to optimize the enzymes for GAP-fill polymerization.4a) Samples were detected using Detection oligo probe (L8748-Cy3); 4b) Samples were inspected for residual signals after stripping the previous detection oligo from RCPs. 4c) Re-probing with GAP fill probe detection oligo (L11524-Cy3) using anchor primers (L11286). Nuclei of the cells are stained with DAPI (blue)